表达猪瘟病毒石门株E2抗原的重组腺病毒构建及免疫性研究

应用PCR从pMD18-T-E2质粒中扩增编码猪瘟病毒(CSFV)E2蛋白的基因片段,定向克隆到重组腺病毒Adeasy-1系统的穿梭质粒pAdTrack-CMV上,采用细菌内同源重组"两步转化法"构建携带CSFVE2基因的重组腺病毒基因组质粒pAdEasy-E2,转染293细胞,成功包装出重组腺病毒pAd-E2,PCR证实E2基因已整合至腺病毒基因组中,Western blot结果检测到重组病毒转染293细胞中E2蛋白的表达。ELISA检测结果表明,pAd-E2能刺激小鼠产生高效价的抗体,也能刺激猪体产生抗体。将pAd-E2通过肌肉和皮下接种途径免疫商品化猪2次,21d后用1000倍TCID50CSFV强毒攻击。pAd-E2免疫组有1/4头死亡,而非重组腺病毒pAd-CMV免疫组和空白对照组全部死亡。该研究为研制猪瘟基因工程疫苗提供了有用的数据。

Construction of the Recombinant Adenovirus Expressing the E2 Gene of Classical Swine Fever Virus Shimen Strain and the Animal Immunity Experiment

The CSFV E2 gene was amplified from the plasmid pMD18-T-E2 by PCR and cloned into the adenoviral shuttle plasmid pAdTrack-CMV.A two-step transformation protocol was employed for the construction of recombinant adenoviral plasmid pAdEasy-E2.The identified recombinant DNA was transfected into 293 cells to package adenovirus.E2 gene was confirmed to be integrated into the genome of recombinant adenovirus by PCR,and the expression of E2 was detected in 293 cells transfected with recombinant adenovirus by Western blot.ELISA results showed that systemic immune responses to CSFV could be induced effectively in mice and pigs by immunization with pAd-E2.pAd-E2 was administered to commercially available pigs through subcutaneous and intramascular routes two times and their susceptibility to CSFV was determined 21 days later by the chanllenge with 1000 TCID50 of CSFV.1/4 of the animals vaccinated with pAd-E2 was dead,and 4/4 of the animals vaccinated with the non-recombinant human adenovirus(pAd-CMV)and 2/2 of the blanks were all dead.