西藏株小反刍兽疫病毒H蛋白的原核表达及其多克隆抗体的制备

【背景】小反刍兽疫是由小反刍兽疫病毒(Peste des petits ruminants virus,PPRV)引起的一种急性、烈性、接触性传染病,严重威胁我国养羊业的发展。【目的】原核表达PPRVH蛋白,并制备其多克隆抗体。【方法】根据GenBank中PPRV西藏株h基因序列,对其进行密码子大肠杆菌偏爱性优化,采用两步PCR法全化学合成全长h基因。将测序验证正确的h基因克隆至原核表达载体pET-28a、pET-30a、pET-32a,转化E. coli BL21(DE3)并利用IPTG诱导H蛋白表达。以经SDS-PAGE割胶纯化的重组H蛋白免疫新西兰大白兔制备抗PPRV H蛋白多克隆抗体。【结果】重组E. coli pET-28a(-30a,-32a)-H]表达的重组H蛋白相对分子质量分别约为70、68和86 kD;诱导7 h时PRRV H蛋白表达量最高,而且主要以包涵体形式表达;重组E.coli(pET-30a-H)表达的H蛋白经SDS-PAGE割胶纯化后免疫新西兰大白兔制备的多抗血清能与表达的重组H蛋白发生特异性反应;ELISA法检测抗体效价在1:6400-1:25600之间。【结论】原核表达了PPRVH蛋白,并制备了高效价的抗H蛋白多克隆抗体,为进一步研究PPRV H蛋白的功能及H蛋白的线性B细胞表位作图奠定了基础。

Prokaryotic expression and polyclonal antibody preparation of peste des petits ruminants virus H protein

Background] Peste des petits ruminants (PPR) is an acute, severe and contagious infectious disease caused by peste des petits ruminants virus (PPRV), which is endangering the sheep/goat-farming in Asian and African countries. Objective] The objective of this study is to express PPRV H protein prokaryotically and to prepare its polyclonal antibodies. Methods] According to the sequence of PPRV Tibet strain deposited in GenBank, h gene was optimized based on the codon usage bias of E. coli and synthesized by using two-step PCR. Then, the sequencing confirmed h gene was inserted into prokaryotic expression vectors pET-28a, pET-30a and pET-32a, respectively. The constructed recombinant plasmids pET-28a(-30a and -32a)-H were then transformed into E. coli BL21(DE3) for expression under the induction of IPTG. The expressed H protein from E. coli BL21(pET-30a-H) were purified by cutting out the aimed band from the SDS-PAGE gel. Polyclonal antibodies were prepared by immunizing New Zealand white rabbits with the purified H protein. Results] The recombinant H protein expressed by E. coli (pET-28a (-30a, -32a)-H), with a relative molecular mass of about 70, 68 and 86 kD, respectively, mainly existed in a form of inclusion body, of which the expression level reached the maximum at 7 h after induction. The polyclonal antibodies prepared by immunizing the rabbits with the purified H protein from E. coli BL21(pET-30a-H) showed specific reactivity with expressed recombinant H protein. The titer values of the serum ranged between 1:6 400 to 1:25 600. Conclusion] PPRV H protein was successfully expressed in prokaryotic cells, and high titer anti-H protein polyclonal antibodies were obtained, which laid a foundation for further research on the function of H protein, and the fine linear B cell epitope mapping of PPRV H protein.