猪瘟病毒E2基因重组鸡痘病毒的构建及免疫保护试验

Construction of the Recombinant Fowlpox Virus Expressing the E2 Gene of Classical Swine Fever Virus and Immunological Protective Experiment

应用PCR从pMD18-T-E2质粒中扩增编码猪瘟病毒(classical swine fever virus,CSFV)E2蛋白的基因片段,定向克隆到重组鸡痘病毒表达载体FPV-P11上,构建出重组鸡痘病毒转移载体FPV-pSY-E2。用脂质体将该质粒转染至鸡痘病毒感染的鸡胚成纤维细胞(CEF)后,通过蓝斑纯化试验筛选出重组鸡痘病毒FV282-CSFV-E2。PCR证实E2基因已整合至鸡痘病毒基因组中。重组鸡痘病毒3次腹腔接种小鼠,ELISA检测血清猪瘟病毒抗体滴度为1:4096。给猪颈部和腹股沟皮下分点注射免疫3次,4周后用CSFV石门强毒株以100LD50接毒量攻击,结果保护率为75%。本试验为猪瘟重组鸡痘病毒活载体疫苗的研制奠定了基础。

The CSFV E2 gene was amplified from the plasmid pMD18-T-E2 by PCR and cloned into the FPV-P1 land FPV-pSY. The identified recombinant DNA was transfected into chicken embryo fibroblasts （CEF）to package Fowlpox virus. E2 gene was confirmed to be integrated into the genome of recombinant Fowlpox virus by PCR. Western blot was employed for detection of the expression of E2 in the chicken embryo fibroblasts infected with recombinant Fowlpox virus . The results of ELISA showed that systemic immune responses to CSFV could be induced effectively after the mice were imnmnized three times with recombinant Fowlpox virus through celiac routes. The titer of CSFV antibody was 1：4096. After immunized three times with recombinant Fowlpox virus, pigs were challenged with 100 LD50 CSFV Shimen strain, and 75% of pigs were protected, which indicated that recombinant Fowlpox virus was efficient. This study built a foundation for the development of CSFV live vector vaccine.