肾型IBVS1基因重组BCG菌株的构建及其免疫原性研究

Construction of recombinant BCG bearing S1 glycoprotein of nephropathogenic IBV and study on its immunogenicity on chickens

摘要:

S1基因是传染性支气管炎病毒(IBV)的主要保护性抗原基因,应用RT-PCR技术特异地扩增嗜肾型传染性支气管炎病毒X株的S1基因,经序列分析证实为S1基因,将其连接到含有人结核分枝杆菌启动子hsp70基因和堪萨斯分枝杆菌α信号肽基因的表达载体pRR3上,从而构建了大肠杆菌-分枝杆菌穿梭表达质粒pR-α-S1,再电转化至BCG中,形成重组菌株rBCG-S1。热激后S1蛋白在耻垢分枝杆菌M.smegmatis mc² 155中高效表达,并且通过ELISA和Western blot检测能够被抗IBVS1蛋白的单克隆抗体识别。将10⁶CFU的重组菌株rBCG-S1皮下注射免疫6周龄的SPF鸡,动物保护试验结果表明重组菌株rBCG-S1对鸡具有一定的免疫保护效果,能够保护SPF鸡抵抗IBVX毒株强毒攻击。血凝抑制试验表明血凝抑制抗体滴度能够显著增加。构建的重组菌株为今后开发新型鸡传染性支气管炎基因工程弱毒疫苗奠定了基础。

Abstract:

Glycoprotein S1 was the major protein to determine infection and immunogenicity of Infectious bronchitis virus（IBV）. The S1 glycoprotein gene of IBV isolates were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) and proved to be S1 gene by sequencing. The E. coli-mycobacterium expression shuttle plasmid pR-α-S1 was constructed by inserting the S1 gene to the pRR3 with human mycobacterium tuberculosis HSP70 promoter and α signal peptide. Then the plasmid pR-α-S1 was introduced into mycobacterium bovis BCG by electroporation to construct a recombinate strain rBCG-S1. The S1 protein could be highly expressed in M. smegmatis mc² 155 when induced by heating and was detected by ELISA and Western blot assays using monoclonal antibody against S1 glycoprotein of IBV. 6 week-old SPF chicken were subcutaneously immunized with 10⁶ cfu rBCG-S1 and each chick was immunized three times at 3 week intervals with the same antigen used for the primary immunization. The protective immunity of rBCG-S1 was identified in vaccinated chickens. Results from the protection test showed the two immunizations with rBCG-S1 could provide protection for chickens from the challenge with virulent nephropathogenic IBV strain X. Haemagglutination inhibition titers were also increased in chickens immunized with the expressed rBCG-S1, and significantly higher titers were detected after challenge. These data indicate that the rBCG-S1 could be used as candidate of a live vector vaccine for NIBV.