H5N8亚型禽流感病毒HA蛋白的原核及真核表达及兔多克隆抗体的制备

Expression and preparation of prokaryotic and eukaryotic HPAIV H5N8 subtype HA gene and rabbit polyclonal antibodies

目的 构建H5N8亚型禽流感病毒(Avian influenza virus, AIV)HA基因的原核和真核表达载体,分别在E.coli Rosetta(DE3)和Sf9中进行表达,并制备兔多克隆抗体。方法 采用RT-PCR扩增H5N8亚型AIV的HA基因,并将其克隆至原核表达载体pTac-His-MBP-PreScission中,再转化至Rosetta(DE3)中,利用IPTG诱导表达。通过SDS-PAGE及蛋白免疫印迹(Western blot, WB)鉴定HA蛋白的表达,并优化IPTG浓度、诱导时间和温度以提高表达效率。将纯化的重组蛋白免疫新西兰大白兔,制备兔抗H5N8亚型AIV的HA蛋白多克隆抗体,通过酶联免疫吸附实验(Enzyme linked immunosorbent assay, ELISA)检测其特异性和效价。同时,将HA基因克隆到真核表达载体pFastBacHTA中,将其瞬时转染Sf9细胞并扩增3代,最后通过WB和间接免疫荧光试验(Indirect immunofluorescence assay, IFA)验证表达产物。结果 成功构建了HA基因的原核重组质粒pTac-HA-DE3和真核重组质粒pFast-HA。经SDS-PAGE及WB验证该蛋白分子质量约110 ku,最佳表达条件为IPTG浓度为0.8 mmol/L、温度37℃和诱导时间为4 h。ELISA检测显示兔多克隆抗体效价为1∶102 400。成功将真核重组质粒pFast-HA转化入DH10Bac感受态细胞中,证明HA基因成功转座。Bacmid-HA转染Sf9细胞后观察到细胞病变,WB和IFA检测表明重组杆状病毒Bac-HA感染的Sf9细胞能够表达HA蛋白。结论 本研究初步探究了H5N8亚型AIV HA蛋白的表达条件及免疫原性,并且具有良好的免疫原性和反应原性,为后续建立ELISA诊断试剂盒及H5N8 AIV疫苗的研制提供了一定的参考价值。

Objective To construct prokaryotic and eukaryotic expression vectors for the HA gene of H5N8 subtype avian influenza virus(AIV),express them in E.coli Rosetta(DE3)and Sf9 cells,respectively,and prepare polyclonal antibodies in rabbits.Methods The HA gene of H5N8 subtype AIV was amplified by RT-PCR and cloned into the prokaryotic expression vector pTac-His-MBP-PreScission,which was then transformed into Rosetta(DE3)cells and induced by IPTG.The expression of the HA protein was identified by SDS-PAGE and Western blot(WB),and the expression efficiency was optimized by varying IPTG concentration,induction time,and temperature.The purified recombinant protein was used to immunize New Zealand rabbits to produce polyclonal antibodies against H5N8 subtype AIV HA protein.The specificity and titer of the antibodies were determined by enzyme-linked immunosorbent assay(ELISA).Additionally,the HA gene was cloned into the eukaryotic expression vector pFastBacHTA,transiently transfected into Sf9 cells and amplified for three generations.The expression of the recombinant protein was verified by WB and indirect immunofluorescence assay(IFA).Results The prokaryotic recombinant plasmid pTac-HA-DE3 and eukaryotic recombinant plasmid pFast-HA were successfully constructed.SDS-PAGE and WB confirmed the molecular weight of the expressed protein to be approximately 110 ku.The optimal expression conditions were determined as an IPTG concentration of 0.8 mmol/L,a temperature of 37℃,and an induction time of 4 hours.ELISA showed that the titer of the rabbit polyclonal antibodies was 1∶102400.Successful transposition of the HA gene was demonstrated by transforming the eukaryotic recombinant plasmid pFast-HA into DH10Bac competent cells.Cell cytopathic effects were observed after transfecting Sf9 cells with Bacmid-HA,and WB and IFA confirmed the expression of HA protein in Bac-HA-infected Sf9 cells.Conclusion This study investigated the expression conditions and immunogenicity of the HA protein of H5N8 subtype AIV,which exhibited good immunogenicity and reactivity.These findings provide valuable references for the development of ELISA diagnostic kits and H5N8 AIV vaccines in future research.