H3N2亚型猪流感病毒NP蛋白的表达及其单克隆抗体的制备
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摘要
猪流感病毒(Swine influenza virus,SIV)是由10种蛋白所构成的球状病毒粒子,其片段5所编码的核蛋白(NP)是保守性结构蛋白,在不同亚型流感病毒中变异率极低。鉴于SIV抗原多变性及其对人类的潜在威胁,NP蛋白单克隆抗体的制备对不同亚型SIV的诊断及NP蛋白结构与功能的研究具有重要的意义。
本研究以携带有特定酶切位点的NP基因上下游引物,以H3N2 SIV cDNA为模板经PCR扩增NP基因,克隆到pMD19-T-simple vector,酶切及测序鉴定正确后构建pET32a-NP表达质粒,转化Rosetta(DE3)进行优化表达,获得分子量约73ku的重组可溶性NP蛋白。该蛋白纯化后经Western blot检测表明可分别与H1N1、H3N2及H9N2亚型SIV鸡阳性血清发生反应,具有良好的反应原性和交叉反应性。同时用此蛋白免疫小鼠制备的抗血清经间接免疫荧光(IFA)检测表明:该血清反过来与H1N1、H3N2及H9N2亚型SIV也可发生特异性反应,进一步证实所表达的NP蛋白同时具有良好的免疫原性。
将重组质粒pMD-NP进行双酶切反应并将NP基因连接到经相同处理的真核表达载体pCAGGS上,经PCR及酶切鉴定获得重组质粒pCAGGS-NP,转染293T细胞,通过IFA和Western blot检测表明NP蛋白在293T细胞中获得了正确表达。
将pCAGGS-NP以100μg/只剂量免疫4-5周龄BALB/c小鼠,按常规方法进行3次免疫后进行加强免疫,3天后取其脾细胞与SP2/0进行融合。融合10天后以原核表达NP蛋白作为筛选抗原,以建立的间接ELISA方法筛选阳性杂交瘤细胞。将筛选到的阳性杂交瘤细胞经3-5次克隆培养后获得1株稳定分泌抗SIV NP蛋白单克隆抗体(MAb)的杂交瘤细胞株(3D7);该单抗具有良好的特异性,经Ig亚类鉴定为IgM,轻链为κ链,其诱导小鼠产生的腹水ELISA效价为1:105;纯化3D7腹水并对其进行亲和力及抗原结合活性的测定,ELISA曲线图显示亲和力常数为1.02×106M-1,IFA结果显示其可与H1N1、H3N2、H9N2 SIV及甲型H1N1流感病毒发生特异性反应,具有良好的交叉反应活性。该MAb的成功制备将为进一步建立SIV的诊断方法以及分析NP蛋白抗原表位等方面的研究奠定基础。
Swine influenza virus (SIV) composed of 10 proteins is spherical virus particles. Its nucleoprotein (NP) encoded by the fifth fragment is conserved structural protein, and low mutation rate in different subtypes of influenza viruses. Given the high variability of SIV antigen and potential threat to humanity, the preparation of monoclonal antibodies against NP protein is important to develop the diagnostic reagents for the different subtypes SIVs and to understand the structure and function of NP protein.
The coding fragment of H3N2 SIV NP gene was amplified using a pair of primers with specific enzyme digestion site by PCR and cloned into plasmid pMD19-Simple-T vector. It was analyzed by restriction enzyme and comfirmed by DNA sequencing, then the NP gene was subcloned into prokaryotic expression vector pET32a. Rosetta ( DE3 ) transformed with recombinant plasmid pET32a-NP was induced by IPTG and the soluble NP protein was expressed effectively,with molecular weight was 73ku.The immune activity of NP protein was confirmed by Western-blot using the specific sera against H1N1、H3N2 and H9N2 subtype SIV. The multi-clonal sera were produced by inoculating the purified NP protein into mice, and then the sera was used to detect H1N1, H3N2 and H9N2 subtype SIV in indirect fluorescence assay (IFA). Above these results of indirect IFA demonstrated that the recombinant protein has good immuogenecity.
Meanwhile, to construct the recombinant plasmid pCAGGS-NP, NP gene was sub-cloned into a eukaryotic expression vector pCAGGS. The recombinant plasmid was transfected into 293T cells, and then the expressed NP protein was confirmed by indirect fluorescence assay (IFA) and Western blot.
BALB/c mice were inoculated with 100μg of the recombinant plasmid pCAGGS-NP. After the third immunization, Splenocytes from the immunized mice were fused with SP2/0 myeloma cells. Positive hybridoma clones were screened by ELISA. After three cycles of subcloning,a monoclonal hybridoma cell line (3D7) against the NP of H3 subtype SIV was generated. The 3D7 MAb has good specificity. The ascites ELISA titer of the MAb was 1: 105 and belonged to IgM subclass,κchain. The relative affinity of the purified MAb against purified H3 SIV was 1.02×106M-1. Furthermore, we also tested the reactivity of the MAb by detecting the H1N1、H3N2 and H9N2 subtype SIVs in IFA. The results demonstrated that the MAb had good cross-reactivity with these SIVs. The preparation of the MAb against NP protein will provide reagents for diagnosis of SIV and analyzing the epitope of NP protein in the future.
本研究以携带有特定酶切位点的NP基因上下游引物,以H3N2 SIV cDNA为模板经PCR扩增NP基因,克隆到pMD19-T-simple vector,酶切及测序鉴定正确后构建pET32a-NP表达质粒,转化Rosetta(DE3)进行优化表达,获得分子量约73ku的重组可溶性NP蛋白。该蛋白纯化后经Western blot检测表明可分别与H1N1、H3N2及H9N2亚型SIV鸡阳性血清发生反应,具有良好的反应原性和交叉反应性。同时用此蛋白免疫小鼠制备的抗血清经间接免疫荧光(IFA)检测表明:该血清反过来与H1N1、H3N2及H9N2亚型SIV也可发生特异性反应,进一步证实所表达的NP蛋白同时具有良好的免疫原性。
将重组质粒pMD-NP进行双酶切反应并将NP基因连接到经相同处理的真核表达载体pCAGGS上,经PCR及酶切鉴定获得重组质粒pCAGGS-NP,转染293T细胞,通过IFA和Western blot检测表明NP蛋白在293T细胞中获得了正确表达。
将pCAGGS-NP以100μg/只剂量免疫4-5周龄BALB/c小鼠,按常规方法进行3次免疫后进行加强免疫,3天后取其脾细胞与SP2/0进行融合。融合10天后以原核表达NP蛋白作为筛选抗原,以建立的间接ELISA方法筛选阳性杂交瘤细胞。将筛选到的阳性杂交瘤细胞经3-5次克隆培养后获得1株稳定分泌抗SIV NP蛋白单克隆抗体(MAb)的杂交瘤细胞株(3D7);该单抗具有良好的特异性,经Ig亚类鉴定为IgM,轻链为κ链,其诱导小鼠产生的腹水ELISA效价为1:105;纯化3D7腹水并对其进行亲和力及抗原结合活性的测定,ELISA曲线图显示亲和力常数为1.02×106M-1,IFA结果显示其可与H1N1、H3N2、H9N2 SIV及甲型H1N1流感病毒发生特异性反应,具有良好的交叉反应活性。该MAb的成功制备将为进一步建立SIV的诊断方法以及分析NP蛋白抗原表位等方面的研究奠定基础。
Swine influenza virus (SIV) composed of 10 proteins is spherical virus particles. Its nucleoprotein (NP) encoded by the fifth fragment is conserved structural protein, and low mutation rate in different subtypes of influenza viruses. Given the high variability of SIV antigen and potential threat to humanity, the preparation of monoclonal antibodies against NP protein is important to develop the diagnostic reagents for the different subtypes SIVs and to understand the structure and function of NP protein.
The coding fragment of H3N2 SIV NP gene was amplified using a pair of primers with specific enzyme digestion site by PCR and cloned into plasmid pMD19-Simple-T vector. It was analyzed by restriction enzyme and comfirmed by DNA sequencing, then the NP gene was subcloned into prokaryotic expression vector pET32a. Rosetta ( DE3 ) transformed with recombinant plasmid pET32a-NP was induced by IPTG and the soluble NP protein was expressed effectively,with molecular weight was 73ku.The immune activity of NP protein was confirmed by Western-blot using the specific sera against H1N1、H3N2 and H9N2 subtype SIV. The multi-clonal sera were produced by inoculating the purified NP protein into mice, and then the sera was used to detect H1N1, H3N2 and H9N2 subtype SIV in indirect fluorescence assay (IFA). Above these results of indirect IFA demonstrated that the recombinant protein has good immuogenecity.
Meanwhile, to construct the recombinant plasmid pCAGGS-NP, NP gene was sub-cloned into a eukaryotic expression vector pCAGGS. The recombinant plasmid was transfected into 293T cells, and then the expressed NP protein was confirmed by indirect fluorescence assay (IFA) and Western blot.
BALB/c mice were inoculated with 100μg of the recombinant plasmid pCAGGS-NP. After the third immunization, Splenocytes from the immunized mice were fused with SP2/0 myeloma cells. Positive hybridoma clones were screened by ELISA. After three cycles of subcloning,a monoclonal hybridoma cell line (3D7) against the NP of H3 subtype SIV was generated. The 3D7 MAb has good specificity. The ascites ELISA titer of the MAb was 1: 105 and belonged to IgM subclass,κchain. The relative affinity of the purified MAb against purified H3 SIV was 1.02×106M-1. Furthermore, we also tested the reactivity of the MAb by detecting the H1N1、H3N2 and H9N2 subtype SIVs in IFA. The results demonstrated that the MAb had good cross-reactivity with these SIVs. The preparation of the MAb against NP protein will provide reagents for diagnosis of SIV and analyzing the epitope of NP protein in the future.
引文
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