【摘要】 gB基因是传染性喉气管炎病毒(ILTV)的主要保护性抗原基因,本试验以ILTV基因组为模板,利用PCR特异扩增出gB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-gB,再电转化至BCG中,形成重组菌株rBCG-gB。热激后gB蛋白在耻垢分枝杆菌M.smegmatismc2155中高效表达,并且通过ELISA和Western blotting检测能够被抗ILTVgB蛋白的单克隆抗体识别。将106CFU的重组菌株rBCG-gB皮下注射免疫3周龄的SPF鸡,分别测定了淋巴细胞指数(SI)、吞噬细胞的吞噬能力以及血清中IL-2和IFN-γ的水平,结果表明重组菌株rBCG-gB作为载体疫苗具有较强的免疫反应。动物保护试验结果表明,重组菌株rBCG-gB对鸡具有一定的免疫保护效果,能够保护SPF鸡抵抗ILTV强毒株的攻击,血凝抑制试验表明血凝抑制抗体滴度能够显著增加。更多还原

【Abstract】 Glycoprotein gB was the major protein to determine infection and immunogenicity of infectious Laryngotracheitis virus(ILTV).The gB glycoprotein gene of ILTV isolates were amplified by polymerase chain reaction(PCR) and proved to be gB gene by sequencing.The E.coli-mycobacterium expression shuttle plasmid pR-α-gB was constructed by inserting the gB gene to the pRR3 with human mycobacterium tuberculosis HSP70 promoter and α signal peptide.Then the plasmid pR-α-gB was introduced into mycobacterium bovis BCG by electroporation to construct a recombinate strain rBCG-gB.The gB 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 gB glycoprotein of ILTV.3 week old SPF chinken were subcutaneously immunized twice at week 0 and 3 with 106 cfu rBCG-gB then the level of lymphocytes index(SI),macrophage activity,cytokine IL-2 and IFN-γ in serum was measured respectively.These results indicated that the recombinant strain rBCG-gB designed as BCG vector vaccine would be able to induce stronger immune response in SPF chicken than in control,vector and BCG groups.6 week-old SPF chicken were subcutaneously immunized with 106 cfu rBCG-gB 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-gB was identified in vaccinated chickens.Results from the protection test were evaluated the two immunizations with rBCG-gB could provide protection for chickens from the challenge with virulent ILTV Heb01strain.Haemagglutination inhibition titers were also increased in chickens immunized with the expressed rBCG-gB,and significantly higher titers were detected after challenge.These data indicate that the rBCG-gB could be used as candidate of a live vector vaccine for ILTV.更多还原