Article(id=1226236830903877849, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1226236828399878330, articleNumber=null, orderNo=null, doi=10.13343/j.cnki.wsxb.20240704, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1730995200000, receivedDateStr=2024-11-08, revisedDate=null, revisedDateStr=null, acceptedDate=1737388800000, acceptedDateStr=2025-01-21, onlineDate=1770287242866, onlineDateStr=2026-02-05, pubDate=1746288000000, pubDateStr=2025-05-04, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1770287242866, onlineIssueDateStr=2026-02-05, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1770287242866, creator=13701087609, updateTime=1770287242866, updator=13701087609, issue=Issue{id=1226236828399878330, tenantId=1146029695717560320, journalId=1192105938417971205, year='2025', volume='65', issue='5', pageStart='1831', pageEnd='2319', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1770287242269, creator=13701087609, updateTime=1770344517883, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1226477059812274835, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1226236828399878330, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1226477059816469140, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1226236828399878330, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=2267, endPage=2279, ext={EN=ArticleExt(id=1226236831189090531, articleId=1226236830903877849, tenantId=1146029695717560320, journalId=1192105938417971205, language=EN, title=Prokaryotic expression and immune effect evaluation of PRRSV nonstructural protein NSP1, columnId=1192149543992045670, journalTitle=Acta Microbiologica Sinica, columnName=Research Article, runingTitle=null, highlight=null, articleAbstract=

[Objective] To express the recombinant nonstructural protein NSP1 of the porcine reproductive and respiratory syndrome virus (PRRSV) strain NADC 30, evaluate its immune effect in mice, and explore the potential value of the nonstructural proteins of PRRSV as vaccine antigens. [Methods] The prokaryotic expression system was used to express NSP1 of NADC30. After purification, the expression and antibody reactivity of NSP1 in vitro were identified by Western blotting. After mice were immunized with NSP1, the levels of cellular and humoral immunity induced by NSP1 were measured. The level of neutralizing antibody induced by NSP1 was evaluated by the virus neutralization assay. [Results] The target gene of NSP1 was connected to the Escherichia coli pET-28a vector for prokaryotic expression. Western blotting identified that the recombinant protein NSP1 was correctly expressed and had antibody reactivity. After mice were immunized with the confirmed recombinant protein NSP1, the levels of interferon (IFN)-γ and tumor necrosis factor (TNF)-α in the spleen lymphocytes of mice were elevated, and cellular immunity was stimulated. At the same time, the recombinant protein NSP1 significantly improved the proliferation of spleen lymphocytes in mice. ELISA results suggested that the level of specific antibodies in the serum rose after immunization. Further analysis of the specific antibody subtypes (IgG2a and IgG1) produced showed that the type of immunity stimulated by recombinant NSP1 was biased to Th2 humoral immunity. In addition, the virus neutralization assay showed that the recombinant protein NSP1 had a good virus neutralization ability, with the neutralization titer of 1:37 on day 28 and 1:31 on day 42, which were significantly higher than those of the PBS control group and had no difference from those of the commercial vaccine group. [Conclusion] The recombinant nonstructural protein NSP1 of PRRSV can stimulate cellular and humoral immunity in mice and has a good virus neutralization ability, which provides a new idea for the development of next-generation PRRSV vaccines.

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E-mail: MA Zhongchen,
CHEN Chuangfu,
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【目的】表达并获取猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus, PRRSV) NADC 30毒株的重组非结构蛋白NSP1,评估其在小鼠体内的免疫效果,并探究PRRSV非结构蛋白作为疫苗抗原的潜在应用价值。【方法】利用原核表达系统表达PRRSV NADC30毒株的NSP1蛋白,纯化后通过Western blotting验证NSP1的体外表达及其抗体反应性;将纯化后的重组NSP1蛋白免疫小鼠,测定其在小鼠体内诱导的细胞免疫和体液免疫水平;通过病毒中和试验评价重组蛋白NSP1诱导的中和抗体水平。【结果】将NSP1目的基因连接至大肠杆菌pET-28a载体并进行原核表达,Western blotting结果显示,重组非结构蛋白NSP1能够正确表达,并且具有抗体反应性。经鉴定正确的重组蛋白NSP1免疫小鼠后,能够提高小鼠脾淋巴细胞中IFN-γ和TNF-α的表达水平,刺激机体产生细胞免疫应答。同时,重组蛋白NSP1还能够显著增强小鼠脾淋巴细胞的增殖能力。ELISA检测结果显示,免疫小鼠血清中的特异性抗体水平升高,进一步分析特异性抗体亚型(IgG2a和IgG1)发现,重组蛋白NSP1刺激机体产生的免疫类型偏向于Th2型体液免疫;病毒中和试验表明,重组蛋白NSP1具有良好的病毒中和能力,在免疫后28 d时中和效价为1:37,42 d时中和效价为1:31,均显著高于PBS对照组,与商品化疫苗组无差异。【结论】PRRSV重组非结构蛋白NSP1免疫小鼠后能够刺激机体产生细胞免疫应答与体液免疫应答,并且具有良好的病毒中和能力,为新型PRRSV疫苗的开发提供了潜在的新思路。

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作者贡献声明

王文星:实验操作,数据收集和处理,论文撰写和修改;李红欢:实验技术指导,协助实验操作与论文修改;顾晓晓:协助实验操作;刘紫威:协助实验操作;吴澳迪:生物学分析;徐明国:生物学分析;马忠臣:论文写作指导与修改;陈创夫:实验设计。

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Virology, 2010, 406(2): 270-279., articleTitle=Porcine reproductive and respiratory syndrome virus non-structural protein 1 suppresses tumor necrosis factor-alpha promoter activation by inhibiting NF-κB and Sp1, refAbstract=null)], funds=[Fund(id=1226592766784877241, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236830903877849, awardId=21322912D, language=EN, fundingSource=Key Research and Development Program of Hebei Province(21322912D), fundOrder=null, country=null), Fund(id=1226592766898123459, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236830903877849, awardId=21322912D, language=CN, fundingSource=河北省重点研发计划(21322912D), fundOrder=null, country=null), Fund(id=1226592766990398151, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236830903877849, awardId=null, language=EN, fundingSource=Program “Tianchi Talent (Young Doctor)” in Xinjiang Uygur Autonomous Region, fundOrder=null, country=null), Fund(id=1226592768374518478, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236830903877849, awardId=null, language=CN, fundingSource=新疆维吾尔自治区“天池英才(青年博士)”计划, fundOrder=null, country=null)], companyList=[AuthorCompany(id=1226592753488933061, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236830903877849, xref=1., ext=[AuthorCompanyExt(id=1226592753505710280, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236830903877849, companyId=1226592753488933061, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1.College of Animal Science and Technology, Shihezi University, Shihezi, Xinjiang, China), AuthorCompanyExt(id=1226592753518293192, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236830903877849, companyId=1226592753488933061, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1.石河子大学 动物科技学院,新疆 石河子)]), AuthorCompany(id=1226592754877247700, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236830903877849, xref=2., ext=[AuthorCompanyExt(id=1226592754881442006, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236830903877849, companyId=1226592754877247700, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2.Collaborative Innovation Center for Sheep Health Breeding and Zoonotic Disease Prevention and Control, Shihezi University, Shihezi, Xinjiang, China), AuthorCompanyExt(id=1226592754889830616, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236830903877849, companyId=1226592754877247700, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2.石河子大学,绵羊健康养殖与人兽共患病防控协同创新中心,新疆 石河子)]), AuthorCompany(id=1226592754998882528, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236830903877849, xref=3., ext=[AuthorCompanyExt(id=1226592755007271135, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236830903877849, companyId=1226592754998882528, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=3.International Joint Research Center for Animal Health, Shihezi University, Shihezi, Xinjiang, China), AuthorCompanyExt(id=1226592755032436961, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236830903877849, companyId=1226592754998882528, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=3.石河子大学,动物健康养殖国际联合研究中心,新疆 石河子)])], figs=[ArticleFig(id=1226592760979960381, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236830903877849, language=EN, label=Figure 1, caption=Structure prediction of NSP1 protein. A: NSP1 protein secondary structure prediction by PSIRPED; B: NSP1 protein secondary structure prediction by SOPMA; C: NSP1α tertiary structure prediction; D: NSP1α tertiary structure Rush diagram; E: NSP1β tertiary structure prediction; F: NSP1β tertiary structure rush diagram; G: Tertiary structure model of NSP1 after the combination of two separate subunits; H: Tertiary structure model of whole protein amino acid sequence prediction; I: Molecular docking simulation., figureFileSmall=w1jBRrRe/rf8vkko5/qm1A==, figureFileBig=c0N60zioFLFIv0koTAlEGw==, tableContent=null), ArticleFig(id=1226592761122566725, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236830903877849, language=CN, label=图1, caption=NSP1蛋白的结构预测。A:PSIPRED预测NSP1蛋白二级结构结果;B:SOPMA预测NSP1蛋白二级结构结果;C:NSP1α三级结构模型;D:NSP1α三级结构拉什图;E:NSP1β三级结构模型;F:NSP1β三级结构拉什图;G:NSP1两个单独亚基结合后的三级结构模型;H:全蛋白氨基酸序列预测的三级结构模型;I:分子对接模拟。, figureFileSmall=w1jBRrRe/rf8vkko5/qm1A==, figureFileBig=c0N60zioFLFIv0koTAlEGw==, tableContent=null), ArticleFig(id=1226592761256784461, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236830903877849, language=EN, label=Figure 2, caption=Double digestion identification of recombinant plasmid. Lane M: 15 kb DNA marker; Lane 1: Double cut plasmid; Lane 2: Uncut plasmid., figureFileSmall=IaGMkJYw5xBKbEyCX8JrGA==, figureFileBig=gHY7eISpBWOrRbT+V0iFLg==, tableContent=null), ArticleFig(id=1226592761382613588, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236830903877849, language=CN, label=图2, caption=重组质粒的双酶切鉴定。泳道M:15 kb DNA marker;泳道1:双酶切质粒;泳道2:未酶切质粒。, figureFileSmall=IaGMkJYw5xBKbEyCX8JrGA==, figureFileBig=gHY7eISpBWOrRbT+V0iFLg==, tableContent=null), ArticleFig(id=1226592761508442715, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236830903877849, language=EN, label=Figure 3, caption=In vitro expression and immunogenicity identification of recombinant protein NSP1. A: Optimization of expression conditions of recombinant protein (lanes 1‒4: The bacterial solution collected at 0 h, 2 h, 4 h and 6 h was induced by 37 ℃, respectively; Lane 5: The bacterial solution collected at 12 h was induced by 16 ℃; B: Reaction of recombinant protein NSP1 with His labeled antibody; C: Reaction of recombinant protein NSP1 with positive serum., figureFileSmall=C++9CwJDEeTw2bDU6GLu1g==, figureFileBig=U9vTmEQQAudiodDntMJ+RQ==, tableContent=null), ArticleFig(id=1226592761638466144, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236830903877849, language=CN, label=图3, caption=重组蛋白NSP1的体外表达与免疫原性鉴定。A:重组蛋白表达条件的优化(泳道1‒4:分别为37 ℃诱导0、2、4、6 h收集的菌液沉淀;泳道5:16 ℃诱导12 h收集的菌液沉淀);B:重组蛋白NSP1与His标签抗体的反应;C:重组蛋白NSP1与阳性血清的反应。, figureFileSmall=C++9CwJDEeTw2bDU6GLu1g==, figureFileBig=U9vTmEQQAudiodDntMJ+RQ==, tableContent=null), ArticleFig(id=1226592761772683876, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236830903877849, language=EN, label=Figure 4, caption=Safety evaluation of mice after immunization. A: The weight change of mice within 7 days after first immunization; B: Changes of body weight in mice within 7 days after second immunization., figureFileSmall=uUyq+z41Ivwye1XXvEhgog==, figureFileBig=r730YNO0xTdr/s5K7gtqsQ==, tableContent=null), ArticleFig(id=1226592761894318697, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236830903877849, language=CN, label=图4, caption=免疫小鼠后的安全性评价。A:小鼠首次免疫后7 d内体重变化;B:小鼠二次免疫后7 d内体重变化。, figureFileSmall=uUyq+z41Ivwye1XXvEhgog==, figureFileBig=r730YNO0xTdr/s5K7gtqsQ==, tableContent=null), ArticleFig(id=1226592761994981999, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236830903877849, language=EN, label=Figure 5, caption=Expression of cytokines in serum and spleen lymphocytes of mice. A: Expression of cytokine mRNA in spleen lymphocytes on day 28; B: Expression of cytokine mRNA level in spleen lymphocytes at day 42 standard curve of IL-10 kit standard product; D: Expression level of IL-10 in serum of mice at day 42. ns: P>0.05; *: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.000 1., figureFileSmall=UoZ0mB6AHmF3QPZNK7Wc0Q==, figureFileBig=1T5ECMwCI2wCwFMVqoSeYg==, tableContent=null), ArticleFig(id=1226592762108228212, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236830903877849, language=CN, label=图5, caption=小鼠血清及脾脏淋巴细胞中各细胞因子的表达。A:28 d脾脏淋巴细胞中细胞因子mRNA水平的表达;B:42 d脾脏淋巴细胞中细胞因子mRNA水平的表达;C:IL-10试剂盒标准品制作的标准曲线;D:42 d时小鼠血清中IL-10表达水平。, figureFileSmall=UoZ0mB6AHmF3QPZNK7Wc0Q==, figureFileBig=1T5ECMwCI2wCwFMVqoSeYg==, tableContent=null), ArticleFig(id=1226592762208891514, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236830903877849, language=EN, label=Figure 6, caption=Lymphocyte proliferation activity and IFN-γ expression. A: Elispot result spot map; B: Expression of IFN-γ at day 28; C: The expression of IFN-γ at day 42; D: Lymphocyte proliferation activity at day 28. ns: P>0.05; *: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.000 1., figureFileSmall=Oq+1VD1QstSet6OWKCm7Hg==, figureFileBig=yOmUpAa8KNxLkW3P9OCfbw==, tableContent=null), ArticleFig(id=1226592762301166209, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236830903877849, language=CN, label=图6, caption=淋巴细胞增殖活性与IFN-γ的表达。A:Elispot结果斑点图;B:28 d时IFN-γ的表达;C:42 d时IFN-γ的表达;D:28 d时淋巴细胞增殖活性。, figureFileSmall=Oq+1VD1QstSet6OWKCm7Hg==, figureFileBig=yOmUpAa8KNxLkW3P9OCfbw==, tableContent=null), ArticleFig(id=1226592762414412426, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236830903877849, language=EN, label=Figure 7, caption=Specific antibody levels of recombinant protein NSP1 in mice. A: Specific antibody IgG level; B: Specific antibody IgG1 level; C: Specific antibody IgG2a level., figureFileSmall=4ghIr21veCfRfWM/sk+v3g==, figureFileBig=VvO+nwKpSuAkmaFl359JQQ==, tableContent=null), ArticleFig(id=1226592763794338452, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236830903877849, language=CN, label=图7, caption=重组蛋白NSP1在小鼠体内的特异性抗体水平。A:特异性抗体IgG水平;B:特异性抗体IgG1水平;C:特异性抗体IgG2a水平。, figureFileSmall=4ghIr21veCfRfWM/sk+v3g==, figureFileBig=VvO+nwKpSuAkmaFl359JQQ==, tableContent=null), ArticleFig(id=1226592765757272727, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236830903877849, language=EN, label=Figure 8, caption=Ratio of specific antibody subtype IgG2a to IgG1., figureFileSmall=rB+An/y0Ag3/2MA8FIXIEw==, figureFileBig=drKmT/0k3ntqihIUCEe4ig==, tableContent=null), ArticleFig(id=1226592766029902492, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236830903877849, language=CN, label=图8, caption=特异性抗体亚型IgG2aIgG1的比值, figureFileSmall=rB+An/y0Ag3/2MA8FIXIEw==, figureFileBig=drKmT/0k3ntqihIUCEe4ig==, tableContent=null), ArticleFig(id=1226592766172508832, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236830903877849, language=EN, label=Figure 9, caption=Neutralizing antibody levels in mice immunized with recombinant protein NSP1. ns: P>0.05;*: P<0.05., figureFileSmall=zp5zG+vSyEtFNpv/0XFZeg==, figureFileBig=0rSARZEKblJosdk8vUx6Xw==, tableContent=null), ArticleFig(id=1226592766294143652, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236830903877849, language=CN, label=图9, caption=重组蛋白NSP1免疫小鼠后的中和抗体水平, figureFileSmall=zp5zG+vSyEtFNpv/0XFZeg==, figureFileBig=0rSARZEKblJosdk8vUx6Xw==, tableContent=null), ArticleFig(id=1226592766440944298, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236830903877849, language=EN, label=Table 1, caption=

Immunization schedule and grouping of mice

, figureFileSmall=null, figureFileBig=null, tableContent=

组别

Group

数目

Number

疫苗

Vaccine

免疫剂量

Immune dose

免疫次数

Immune frequency

免疫周期

Immune cycle

112NSP150 μg+50 μL20 d, 14 d
212PBS100 μL20 d, 14 d
312Vaccines100 μL20 d, 14 d
), ArticleFig(id=1226592766549996208, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226236830903877849, language=CN, label=表1, caption=

小鼠免疫计划及分组情况

, figureFileSmall=null, figureFileBig=null, tableContent=

组别

Group

数目

Number

疫苗

Vaccine

免疫剂量

Immune dose

免疫次数

Immune frequency

免疫周期

Immune cycle

112NSP150 μg+50 μL20 d, 14 d
212PBS100 μL20 d, 14 d
312Vaccines100 μL20 d, 14 d
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PRRSV重组非结构蛋白NSP1表达及免疫效果评价
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王文星 1 , 李红欢 1 , 顾晓晓 1 , 刘紫威 1 , 吴澳迪 1 , 徐明国 1 , 马忠臣 1, 2, 3 , 陈创夫 1, 2, 3
微生物学报 | 研究报告 2025,65(5): 2267-2279
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微生物学报 | 研究报告 2025, 65(5): 2267-2279
PRRSV重组非结构蛋白NSP1表达及免疫效果评价
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王文星1, 李红欢1, 顾晓晓1, 刘紫威1, 吴澳迪1, 徐明国1, 马忠臣1, 2, 3 , 陈创夫1, 2, 3
作者信息
  • 1.石河子大学 动物科技学院,新疆 石河子
  • 2.石河子大学,绵羊健康养殖与人兽共患病防控协同创新中心,新疆 石河子
  • 3.石河子大学,动物健康养殖国际联合研究中心,新疆 石河子
Prokaryotic expression and immune effect evaluation of PRRSV nonstructural protein NSP1
Wenxing WANG1, Honghuan LI1, Xiaoxiao GU1, Ziwei LIU1, Aodi WU1, Mingguo XU1, Zhongchen MA1, 2, 3 , Chuangfu CHEN1, 2, 3
Affiliations
  • 1.College of Animal Science and Technology, Shihezi University, Shihezi, Xinjiang, China
  • 2.Collaborative Innovation Center for Sheep Health Breeding and Zoonotic Disease Prevention and Control, Shihezi University, Shihezi, Xinjiang, China
  • 3.International Joint Research Center for Animal Health, Shihezi University, Shihezi, Xinjiang, China
出版时间: 2025-05-04 doi: 10.13343/j.cnki.wsxb.20240704
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【目的】表达并获取猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus, PRRSV) NADC 30毒株的重组非结构蛋白NSP1,评估其在小鼠体内的免疫效果,并探究PRRSV非结构蛋白作为疫苗抗原的潜在应用价值。【方法】利用原核表达系统表达PRRSV NADC30毒株的NSP1蛋白,纯化后通过Western blotting验证NSP1的体外表达及其抗体反应性;将纯化后的重组NSP1蛋白免疫小鼠,测定其在小鼠体内诱导的细胞免疫和体液免疫水平;通过病毒中和试验评价重组蛋白NSP1诱导的中和抗体水平。【结果】将NSP1目的基因连接至大肠杆菌pET-28a载体并进行原核表达,Western blotting结果显示,重组非结构蛋白NSP1能够正确表达,并且具有抗体反应性。经鉴定正确的重组蛋白NSP1免疫小鼠后,能够提高小鼠脾淋巴细胞中IFN-γ和TNF-α的表达水平,刺激机体产生细胞免疫应答。同时,重组蛋白NSP1还能够显著增强小鼠脾淋巴细胞的增殖能力。ELISA检测结果显示,免疫小鼠血清中的特异性抗体水平升高,进一步分析特异性抗体亚型(IgG2a和IgG1)发现,重组蛋白NSP1刺激机体产生的免疫类型偏向于Th2型体液免疫;病毒中和试验表明,重组蛋白NSP1具有良好的病毒中和能力,在免疫后28 d时中和效价为1:37,42 d时中和效价为1:31,均显著高于PBS对照组,与商品化疫苗组无差异。【结论】PRRSV重组非结构蛋白NSP1免疫小鼠后能够刺激机体产生细胞免疫应答与体液免疫应答,并且具有良好的病毒中和能力,为新型PRRSV疫苗的开发提供了潜在的新思路。

猪繁殖与呼吸综合征病毒  /  重组蛋白NSP1  /  免疫评价  /  疫苗

[Objective] To express the recombinant nonstructural protein NSP1 of the porcine reproductive and respiratory syndrome virus (PRRSV) strain NADC 30, evaluate its immune effect in mice, and explore the potential value of the nonstructural proteins of PRRSV as vaccine antigens. [Methods] The prokaryotic expression system was used to express NSP1 of NADC30. After purification, the expression and antibody reactivity of NSP1 in vitro were identified by Western blotting. After mice were immunized with NSP1, the levels of cellular and humoral immunity induced by NSP1 were measured. The level of neutralizing antibody induced by NSP1 was evaluated by the virus neutralization assay. [Results] The target gene of NSP1 was connected to the Escherichia coli pET-28a vector for prokaryotic expression. Western blotting identified that the recombinant protein NSP1 was correctly expressed and had antibody reactivity. After mice were immunized with the confirmed recombinant protein NSP1, the levels of interferon (IFN)-γ and tumor necrosis factor (TNF)-α in the spleen lymphocytes of mice were elevated, and cellular immunity was stimulated. At the same time, the recombinant protein NSP1 significantly improved the proliferation of spleen lymphocytes in mice. ELISA results suggested that the level of specific antibodies in the serum rose after immunization. Further analysis of the specific antibody subtypes (IgG2a and IgG1) produced showed that the type of immunity stimulated by recombinant NSP1 was biased to Th2 humoral immunity. In addition, the virus neutralization assay showed that the recombinant protein NSP1 had a good virus neutralization ability, with the neutralization titer of 1:37 on day 28 and 1:31 on day 42, which were significantly higher than those of the PBS control group and had no difference from those of the commercial vaccine group. [Conclusion] The recombinant nonstructural protein NSP1 of PRRSV can stimulate cellular and humoral immunity in mice and has a good virus neutralization ability, which provides a new idea for the development of next-generation PRRSV vaccines.

porcine reproductive and respiratory syndrome virus  /  recombinant protein NSP1  /  immune evaluation  /  vaccine
王文星, 李红欢, 顾晓晓, 刘紫威, 吴澳迪, 徐明国, 马忠臣, 陈创夫. PRRSV重组非结构蛋白NSP1表达及免疫效果评价. 微生物学报, 2025 , 65 (5) : 2267 -2279 . DOI: 10.13343/j.cnki.wsxb.20240704
Wenxing WANG, Honghuan LI, Xiaoxiao GU, Ziwei LIU, Aodi WU, Mingguo XU, Zhongchen MA, Chuangfu CHEN. Prokaryotic expression and immune effect evaluation of PRRSV nonstructural protein NSP1[J]. Acta Microbiologica Sinica, 2025 , 65 (5) : 2267 -2279 . DOI: 10.13343/j.cnki.wsxb.20240704
猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus, PRRSV)感染会引发猪繁殖与呼吸综合征(porcine reproductive and respiratory syndrome, PRRS),俗称猪“蓝耳病”,是全球常见的猪病之一,其临床特征主要表现为妊娠母猪流产,产死胎、木乃伊胎,以及仔猪呼吸综合征等,给全球养猪业带来了巨大的经济损失。PRRSV的持续重组和突变使其演化出多种机制来规避宿主的天然免疫反应[1],破坏宿主防御系统,从而为其自身生存提供有利条件。自1996年在中国首次发现PRRSV以来,已经出现了多种遗传差异显著的PRRSV毒株,这使得疾病的预防和控制变得更加复杂[2]。PRRSV NADC 30-Like是国内近年来逐渐流行的新毒株,它与美国的NADC30毒株高度同源,被认为是由NADC30毒株变异演化而来。
PRRSV病毒的基因组全长约15 kb,包含10个或至少8个开放阅读框(open reading frame, ORF)。其中,ORF2‒ORF7负责编码病毒的结构蛋白(N、M、GP2-GP5和E),而PRRSV的复制酶基因由ORF1a和ORF1b组成,复制酶基因占据了整个病毒基因组约3/4的长度,这2个区域分别编码了2个复制酶相关的多蛋白pp1a和pp1ab,这些多蛋白经过加工后会形成15种非结构蛋白(nonstructural proteins, NSPs),这些蛋白对于病毒RNA的合成以及对抗宿主的抗病毒免疫反应至关重要[3-6]。NSP1是一个多功能蛋白,包含2个木瓜样半胱氨酸蛋白酶(PCPα和PCPβ)以及1个锌指结构基序[7]。PCPα和PCPβ分别负责NSP1α和NSP1β的自我蛋白水解释放[8]。NSP1α和NSP1β这2个亚基均定位于细胞核中,这暗示它们可能具有调节细胞功能的潜力[9]。NSP1能够被宿主免疫系统识别,并触发具有中和能力的抗体反应[10]。NSP1蛋白含有高度抗原区域,并且在不同的PRRSV毒株中展现出良好的保守性[11]。Johnson等[12]研究表明,NSP1能够诱导疫苗和野外分离物产生强大且快速的交叉抗体反应。这些研究结果表明,NSP1可能是PRRSV主要的交叉反应抗原,提示针对NSP1的免疫应答在抗PRRSV感染中发挥着关键作用。然而,在国内关于NSP1作为亚单位疫苗抗原的免疫效果尚未见报道。
目前,尚未研制出有效的PRRSV疫苗。尽管已经尝试了DNA疫苗、亚单位疫苗和病毒载体疫苗,但它们作为改良活病毒(modified live virus, MLV)疫苗的替代品,其潜力尚不确定[13]。已经证实,PRRSV NSPs在调节宿主对PRRSV感染的先天免疫应答中扮演了多种角色[9]。NSPs在病毒粒子结构蛋白的加工和成熟中起到了关键作用,因此,针对PRRSV NSPs的早期和持久的免疫反应可能是实现有效控制PRRSV感染的关键。本研究通过原核表达系统表达、纯化了PRRSV NSP1重组蛋白,并对该重组蛋白在小鼠体内的免疫效果进行了初步评价,旨在为新型PRRSV疫苗的开发提供数据支持。
PRRSV XJSW-2021株(GenBank登录号为OR247780.1)和Marc-145细胞由本实验室保存。SPF级6周龄BALB/c小鼠购自河南省实验动物中心。动物实验经石河子大学生物伦理委员会批准(审批号:A2023-246)。
小鼠ELISA IL-10试剂盒,Valukine公司;小鼠Elispot IFN-γ试剂盒,Mabtech公司;小鼠脾脏淋巴细胞分离试剂盒,天津市灏洋生物制品科技有限责任公司;RNA提取试剂盒,北京全式金生物技术股份有限公司;反转录试剂盒,江苏康为世纪生物科技股份有限公司;HRP标记的羊抗鼠特异性抗体IgG、IgG1、IgG2a,武汉三鹰生物技术有限公司;Pierce BCA蛋白检测试剂盒,赛默飞世尔科技公司;质粒小量提取试剂盒和CCK8试剂盒,北京索莱宝科技有限公司;PRRSV抗体阳性血清由本实验室保存。
根据NCBI (https://www.ncbi.nlm.nih.gov/)的PRRSV NADC30毒株NSP1的氨基酸序列(GenBank登录号为QBG05658.1)用PSIPRED Workbench v4.0和NPS@: SOPMA secondary structure prediction-NPSA-Lyon-France进行蛋白二级结构预测,再利用Untitled Project|Modelsy与https://zhanggroup.org/D-I-TASSER/构建蛋白三级结构模型。利用PyMOL软件对NSP1α与NSP1β进行分子对接模拟。
对NSP1的基因序列进行密码子优化,并在基因上游和下游分别加入BamH I和Xho I酶切位点,将目的基因与大肠杆菌pET-28a载体进行连接。重组质粒由北京擎科生物科技股份有限公司合成。
将合成的NSP1重组质粒进行双酶切鉴定,酶切体系:质粒10 μL,ddH2O 6 μL,Buffer 2 μL,酶(浓度为50 U/μL)各1 μL,37 ℃水浴酶切4 h。酶切产物用1%琼脂糖凝胶进行电泳。对酶切正确的质粒进行胶回收后送杭州有康生物科技有限公司测序。
将鉴定正确的重组质粒通过热激法转化BL21(DE3)感受态细胞。将转化产物涂布于含有硫酸卡那霉素(K)抗性的LB固体培养基上,于37 ℃恒温培养箱中培养过夜。第2天挑取单菌落37 ℃、170 r/min振荡培养12 h。将培养好的菌液按菌液与50%甘油1:1的比例保存于甘油管中,置于-20 ℃冰箱冻存。
将菌液分别置于37 ℃、170 r/min摇床上诱导0、2、4、6 h时,分别收集1 mL菌液沉淀;16 ℃、170 r/min摇床上进行低温诱导12 h时收集1 mL菌液沉淀,菌液沉淀进行SDS-PAGE电泳,确定最佳诱导时间和最佳诱导温度。
根据重组蛋白的最佳诱导条件进行大量表达。收集菌液沉淀进行超声破碎(功率35 W,冰浴,工作5 s,停5 s,超声30 min)。破碎后8 000 r/min离心30 min后收集上清与沉淀,通过SDS-PAGE确定重组蛋白的表达形式后,利用镍柱对蛋白进行纯化。纯化后的蛋白装入透析袋进行梯度透析,并检测蛋白浓度。纯化后的蛋白置于-20 ℃冰箱保存。
将重组蛋白进行SDS-PAGE,电泳结束后进行转膜。转膜结束后,使用5%脱脂奶粉封闭液在4 ℃过夜封闭。随后在室温下水平摇床90 r/min孵育一抗2 h,洗膜5次,室温摇床90 r/min孵育二抗1 h,洗膜5次。在暗室中,膜上滴加显影液进行显影。
将纯化好的蛋白按照表1免疫方案进行免疫。免疫方式为小鼠背部皮下多点注射,免疫前对小鼠进行断尾采血作为免疫前阴性对照,免疫后每7 d进行断尾采血。商品化灭活疫苗兰立定(经典毒株CH-1a)作为疫苗组对照。
提前用小鼠28 d血清进行棋盘法筛选间接ELISA测定特异性抗体的反应条件。按照筛选条件分别进行过夜包被蛋白、5%脱脂奶粉封闭、一抗孵育、二抗孵育、显色、终止。终止显色5 min内用酶标仪测量OD450处的吸光度值。
在首次免疫小鼠后的第28天和第42天,无菌分离小鼠的脾脏淋巴细胞。将分离出的脾脏淋巴细胞以每孔1×106个细胞的密度接种到预先包被有IFN-γ的96孔板上,随后进行刺激并置于细胞培养箱中培养48 h。之后弃去培养液,对板进行5次洗涤。按照Elispot IFN-γ试剂盒的说明书进行操作,加入检测抗体并室温孵育2 h,再次洗涤5次。加入链霉亲和素并室温孵育1 h,再次洗涤5次。加入过滤后的底物溶液进行斑点显色,使用大量自来水冲洗以终止显色反应。最后,将平板晾干,并用铝箔包裹后送北京达科为生物技术有限公司进行读板。
将淋巴细胞按照每孔1×105个细胞铺在96孔板上,进行抗原刺激后置于细胞培养箱中培养。加入CCK溶液4 h后用酶标仪读取OD450处的吸光度值,计算刺激指数(stimulus index, SI)如公式(1)所示。
SI刺激指数=(刺激原刺激孔-无刺激对照孔)/(非特异性刺激原刺激孔-无刺激对照孔)
将分离的脾淋巴细胞铺在24孔细胞培养板上,每种细胞因子设置抗原刺激组和未刺激组对照,抗原刺激后24孔板于细胞培养箱培养24 h后收样,提取RNA,测浓度后反转录为cDNA,将cDNA作为模板进行荧光定量PCR检测各细胞因子mRNA水平的表达。
用商品化的小鼠IL-10 ELISA试剂盒测定小鼠血清中的IL-10表达量,按照试剂盒说明书操作,利用标准品制作标准曲线,然后计算样品吸光度值对应的IL-10表达量。
预先测定病毒(毒株为PRRSV XJSW-2021)的TCID50,大量培养Marc-145细胞。将收集的28 d与42 d小鼠血清56 ℃水浴30 min热灭活,经倍比稀释的血清与病毒相互作用2 h (接毒量为200 TCID50),平移至96孔细胞培养板上,与细胞作用1 h后换成含1%胎牛血清的DMEM维持液,连续1周观察细胞病变,以能保护50%细胞不被病毒感染的判定为中和抗体阳性。采用Reed-Muench法计算中和抗体(neutralizing antibody, NA)滴度。
试验数据采用GraphPad Prism 9软件进行双因素方差分析(two-way ANOVA),采用SD检验法进行组间多重比较。P>0.05时,视为差异无统计学意义(ns);P<0.05为差异有统计学意义(*:P<0.05;**:P<0.01;***:P<0.001;****:P<0.000 1)。
通过PSIPRED和SOPMA在线工具对NSP1蛋白进行二级结构分析,预测结果显示该蛋白包含4种二级结构:α螺旋占整个蛋白的20.60%,β折叠占5.76%,无规则卷曲占53.93%,延伸链占19.63% (图1A1B)。三级结构预测发现,NSP1蛋白可在第180位氨基酸处自裂解,形成NSP1α (图1C)和NSP1β (图1E) 2个亚基。NSP1α的三级结构模型拉什图评分为91.00% (图1D),NSP1β的三级结构模型的拉什图评分为91.91% (图1F),表明所构建的三级结构模型是可信的[落在允许区(深色)与最大允许区(浅色)的氨基酸残基占整个蛋白质的比例高于90.00%则认为该模型的构象符合立体化学的规则]。使用I-TASSER对NSP1的2个亚基序列进行单独建模后连接,发现2个亚基结合后的结构(图1G)与基于全蛋白氨基酸序列预测的三级结构模型(图1H) 存在差异,通过PyMOL软件进行分子对接模拟(图1I),发现2个亚基并非简单连接,而是在氢键作用下发生了氨基酸错位结合,这种结合方式与结合位点的改变可能导致蛋白质功能的变化。
将合成的NSP1重组质粒进行双酶切鉴定,结果显示酶切后的质粒得到2条条带,大小分别为5 369 bp和1 158 bp,与预期的目的条带大小相符(图2)。对酶切鉴定正确的条带进行胶回收后送测序,测序结果与目的基因比对匹配度为100%,表明已成功构建出正确的重组质粒。
通过SDS-PAGE分析了在不同诱导条件下获得的菌液沉淀。电泳结果显示,重组蛋白NSP1在37 ℃诱导6 h的表达量最高,因此确定37 ℃诱导6 h为重组蛋白NSP1的最佳诱导条件(图3A)。在后续实验中以此条件进行了大量诱导表达。为了验证纯化后的重组蛋白NSP1在体外的正确表达及其抗体反应性,进行了Western blotting实验。结果显示,重组蛋白NSP1能够与His标签抗体(图3B)以及PRRS阳性血清(图3C)特异性结合,并在约42 kDa处出现特异性条带,这与预期的重组蛋白NSP1分子量相符。这表明重组蛋白NSP1不仅能在体外正确表达,还具有良好的免疫原性。
连续记录小鼠每次免疫后7 d内的体重变化,如图4所示,小鼠在首次免疫后7 d内和二次免疫后7 d内均未出现体重下降现象,整个免疫期间小鼠体重保持稳定,且免疫后小鼠的精神状态与饮食状况良好,无异常行为表现。
在首次免疫后第28天与第42天时分离小鼠的脾脏淋巴细胞,并通过实时荧光定量PCR检测脾脏淋巴细胞中细胞因子mRNA的表达水平。在第28天时,重组蛋白NSP1组和疫苗组均能显著地增加IL-6与TNF-α的表达,且重组蛋白NSP1组IL-6与TNF-α的表达量比疫苗组IL-6与TNF-α的表达量显著性增高(P<0.000 1)。然而,二者IL-4与IL-10的表达量与PBS对照组相比均无显著性差异(P>0.05)。在42天时,同样观察到重组蛋白NSP1组能显著地增加IL-6与TNF-α的表达(P<0.000 1),而IL-4与IL-10的表达与PBS对照组相比无显著性差异(P>0.05) (图5A5B)。使用ELISA试剂盒检测小鼠第42天血清中IL-10的表达量,根据标准品制作的标准曲线(图5C),结果显示重组蛋白NSP1组和疫苗组血清中IL-10的表达量与PBS对照组相比均无显著性差异(P>0.05) (图5D)。
为了进一步评估重组蛋白NSP1在小鼠体内引发的细胞免疫水平。在首次免疫小鼠后的第28天和第42天采用Elispot技术检测了小鼠脾脏淋巴细胞中IFN-γ的分泌情况。结果表明,重组蛋白NSP1在第28天就能显著地增加小鼠脾淋巴细胞分泌IFN-γ的细胞数量(P<0.05),并且到第42天时差异性变得更加明显(P<0.000 1) (图6A)。相比之下,疫苗组免疫小鼠后,在第28天时小鼠脾淋巴细胞分泌IFN-γ的水平与PBS对照组相比无显著性差异(P>0.05) (图6B),直到第42天时才观察到疫苗组显著地增加了小鼠脾淋巴细胞中IFN-γ的表达(P<0.000 1) (图6C)。淋巴细胞增殖实验结果表明,在第28天时重组蛋白NSP1与疫苗组均显著地提高了淋巴细胞的增殖能力(图6D)。上述结果表明,重组蛋白NSP1能够增强小鼠的细胞免疫水平。
通过间接ELISA技术检测特异性抗体水平,进一步评估重组蛋白NSP1在小鼠体内引发的体液免疫效果。结果表明,重组蛋白免疫小鼠后,引发的特异性抗体水平整体呈上升趋势,在第21天时特异性抗体水平开始有所升高,并持续升高至第42天,而疫苗组与PBS组在免疫后的整个周期内并未观察到明显的抗体水平变化(图7A)。同时,检测了特异性抗体亚型IgG1与IgG2a的表达情况,观察到IgG1在首次免疫后第21天开始呈现上升趋势,一直持续到第42天(图7B)。IgG2a在第35天时才开始上升(图7C)。特异性抗体的检测结果表明,重组蛋白NSP1能够在小鼠体内引发高水平的体液免疫。
为了确定重组蛋白在小鼠体内引起的免疫偏向性,进一步分析了小鼠血清中的特异性抗体亚型。结果发现,在免疫小鼠后小鼠体内的特异性抗体亚型IgG2a与IgG1的比值均小于1,说明重组蛋白NSP1在小鼠体内产生的特异性IgG更偏向于亚型IgG1,即重组蛋白NSP1在小鼠体内引起的免疫偏向于Th2型免疫(图8)。
病毒中和试验结果表明,重组蛋白NSP1在首次免疫后的第28天时就已经产生中和抗体,其抗体滴度为1:37,且这一中和抗体水平显著高于PBS对照组(P<0.05),疫苗组的中和抗体滴度为1:39,但重组蛋白NSP1组与疫苗组的中和抗体水平之间无显著差异(P>0.05)。第42天时重组蛋白NSP1的中和抗体滴度略有下降,为1:31 (图9)。
PRRSV感染引发的免疫抑制现象,涵盖了先天免疫反应的减弱以及获得性免疫反应的不良诱导,特别是中和抗体的产生延迟[14-15]。以往的研究多聚焦于PRRSV结构蛋白(例如N或GP5)的免疫反应,但这些蛋白的高度变异性阻碍了我们找到针对该病原体的有效策略。因此,开发新型疫苗对于控制PRRS疫情显得尤为关键[16-17]。PRRSV的NSPs被认为在病毒复制、转录以及宿主先天免疫反应的调节和逃避中扮演了重要角色。在这些NSPs中,NSP1是动脉病毒感染过程中首个合成的病毒蛋白,它在病毒基因组复制中发挥作用,并被认为是迄今为止调节宿主细胞免疫反应最为有效的蛋白[18]。目前,国内尚无关于PRRSV NADC30毒株非结构蛋白NSP1作为亚单位疫苗抗原的研究报道。本研究首先对PRRSV NADC30毒株的NSP1蛋白进行了二级和三级结构的预测,随后将NSP1基因与大肠杆菌pET-28a载体连接并进行原核表达,最后对重组蛋白NSP1在小鼠体内的免疫效果进行了初步评估。
经过重组蛋白NSP1免疫处理后,小鼠体内能够产生Th1型细胞免疫和Th2型体液免疫反应。其中,IFN-γ和TNF-α是Th1型细胞因子的主要代表,而IL-4与IL-10则是Th2型细胞因子的典型成员。IL-6作为B细胞的刺激因子,能够诱导抗体参与免疫调节[19-20]。本研究结果表明,重组蛋白NSP1在免疫小鼠第28天与第42天时均可以显著地提高小鼠脾脏淋巴细胞中IL-6与TNF-α mRNA水平的表达,但对IL-4与IL-10的表达水平未产生明显影响。同时,Elispot结果表明重组蛋白NSP1免疫后刺激小鼠脾脏淋巴细胞分泌的IFN-γ显著高于PBS对照组,表明重组蛋白NSP1免疫后可以促进机体产生细胞免疫应答。
最新研究揭示,NSP1的2个亚基(NSP1α和NSP1β)均具备抑制I型干扰素产生的能力[2,9,18],特别是NSP1β,它作为毒力因子,能够抑制宿主干扰素反应的多个环节[21]。然而,一些研究者提出,动脉病毒NSP1的亚基在细胞核内的作用可能是多变的,其生物学功能与细胞内的分布紧密相关[9]。本研究的结果表明,完整表达NSP1蛋白能够诱导IFN-γ和TNF-α的表达,从而促进机体的细胞免疫应答。这一现象可能与2个亚基之间的相互作用有关,其具体的作用机制尚需进一步探究。同时,尽管有研究指出NSP1α和NSP1β均参与了TNF-α启动子的抑制[22],但关于PRRSV诱导TNF-α表达的机制仍存在争议。Han等[18]研究显示,TNF-α的表达具有毒株依赖性,与PRRSV的经典毒株相比,HP-PRRSV导致的TNF-α表达水平较低。因此,本研究中观察到的TNF-α上调可能与PRRSV NADC30毒株的基因序列或其致病性有关。综上所述,本研究结果证实了NSP1的重组表达能够激发小鼠的免疫反应,为NSP1作为保护性抗原的进一步研究提供了有力支持。
对于由多个亚基组成的蛋白质,亚基之间的相互作用和组装方式对蛋白质的功能至关重要。亚基结合位点发生改变或亚基之间的相互作用被破坏,可能会导致蛋白质的四级结构解体或改变,从而影响其功能。通过对NSP1蛋白及其亚基分别进行三级结构建模,并对2个亚基进行分子对接模拟,发现NSP1免疫功能发生改变可能与2个亚基之间的结合方式发生改变有关。然而,对蛋白三级结构进行预测只能作为一种猜测与预想,我们目前的研究只能确定NSP1蛋白的免疫效果,但其亚基在机体内具体发生了哪些作用或改变仍需进行深入研究。
Johnson等[12]研究表明,NSP1可以在猪体内引起高且持续的抗体反应。为了进一步确定重组蛋白NSP1是否能够在小鼠体内产生体液免疫,本研究检测了免疫后小鼠血清中的抗体水平。结果表明重组蛋白NSP1在首次免疫后第21天时,血清中特异性抗体水平开始上升,并且到42天一直呈上升趋势。Th1型细胞和Th2型细胞可分别促进IgG2a和IgG1的产生。对特异性抗体亚型进一步分析发现,小鼠血清中IgG1抗体水平显著升高,且IgG2a/IgG1的比值在免疫后一直小于1,即免疫后可以引起小鼠产生强烈的Th2型体液免疫,进一步证明重组蛋白NSP1可以诱导机体产生体液免疫应答。
本研究的病毒中和试验结果表明,经过重组蛋白NSP1免疫后,小鼠体内能够产生中和抗体。在28天时中和抗体的效价达到1:37,而到了42天效价为1:31,这2个数值均显著高于PBS对照组,并且与商品化疫苗组之间无显著性差异。这说明重组蛋白NSP1免疫能够诱导小鼠产生高水平的特异性抗体和中和抗体,显示出良好的免疫原性。因此,非结构蛋白NSP1可被视为PRRSV亚单位疫苗的潜在候选抗原。
综合以上研究结果,本研究对PRRSV NADC30毒株的NSP1蛋白进行了结构预测,并通过原核表达系统成功表达了重组蛋白NSP1。免疫小鼠后,发现重组蛋白NSP1能够激发机体产生细胞免疫和体液免疫反应,导致小鼠体内产生较高水平的特异性抗体和中和抗体。因此,重组蛋白NSP1被认为是制备亚单位疫苗的理想候选抗原,为PRRSV新型疫苗的研发提供了新的数据支持。
  • 河北省重点研发计划(21322912D)
  • 新疆维吾尔自治区“天池英才(青年博士)”计划
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2025年第65卷第5期
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doi: 10.13343/j.cnki.wsxb.20240704
  • 接收时间:2024-11-08
  • 首发时间:2026-02-05
  • 出版时间:2025-05-04
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  • 收稿日期:2024-11-08
  • 录用日期:2025-01-21
基金
Key Research and Development Program of Hebei Province(21322912D)
河北省重点研发计划(21322912D)
Program “Tianchi Talent (Young Doctor)” in Xinjiang Uygur Autonomous Region
新疆维吾尔自治区“天池英才(青年博士)”计划
作者信息
    1.石河子大学 动物科技学院,新疆 石河子
    2.石河子大学,绵羊健康养殖与人兽共患病防控协同创新中心,新疆 石河子
    3.石河子大学,动物健康养殖国际联合研究中心,新疆 石河子
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2种不同金属材料的力学参数

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属数
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genus
种数
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species
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Percentage of
total species (%)

Genus
种数
Number of
species
占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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