融合蛋白C3d提高新城疫灭活苗的免疫原性

Increased immunogenicity by conjugation of recombinant complement C3d to inactived NDV vaccine

补体C3d是补体系统(complement system,CS)中补体C3的最终裂解产物。本试验利用RT-PCR技术从AA肉鸡肝脏总RNA中扩增C3d cDNA,测序证实为C3d基因。然后将其连接至表达载体pET-32a(+),转化E.coliBL21(DE3),1%IPTG诱导表达。SDS-PAGE、Western-blotting证实目的蛋白为C3d。亲和层析分离C3d融合蛋白,与纯化新城疫病毒(Lasota株)经戊二醛连接后免疫SPF鸡。血凝抑制试验(HI)检测新城疫抗体水平,MTT法检测胸腺T淋巴细胞转化率。结果表明C3d融合蛋白能够提高特异性抗体水平以及细胞免疫水平,本研究为进一步研究、利用鸡补体C3d奠定了基础。

C3d is the final split product of C3. The C3d eDNA was cloned from the liver of AA chickens through RT-PCR. C3d eDNA was proved to be the C3 d gene of chicken by sequencing. Then, the gene was cloned into pET-32a（ ＋ ）, and transformed the recombinant plasmid into BL21 （ DE3 ） and induced with 1.0 mmol/L IPTG for gene expression. The expressed product was identified by SDS-PAGE and Westernblotting,it was approved that the recombinant protein was recombinant C3d. It was isolated and purified by the method of affinity chromatography. The protein conjugation to inactive NDV（Lasota strain）by glutaual was injected to the SPF chicken, monitored The dynamic changes of T lymphocyte proliferative responses in thymus and the kinetic changes of antibody of immunized chickens were by MTT method and HI methed. It was proved that the recombinant C3d protein could enhance both the humoral and cellular immune response of the inactive NDV vaccine. This report constituted a solid foundation for further researches of chicken C3d.