猪卵透明带3α重组腺病毒的构建及纯化

目的构建猪卵透明带3α(pZP3α)重组腺病毒,并包装与纯化病毒,为研究猪ZP3α重组腺病毒避孕疫苗奠定基础。方法将pZP3α基因克隆至穿梭质粒pShuttle,重组穿梭质粒pSshuttle-pZP3α再与腺病毒骨架质粒pAdEasy-5共转化大肠杆菌BJ5183,筛选重组腺病毒质粒pAd-pZP3α,脂质体法转染HEK293A细胞,包装并扩增病毒至第4代。收集重组病毒颗粒,经两轮氯化铯密度梯度离心纯化,紫外吸收法测定总病毒颗粒数(VP),并计算病毒的颗粒性滴度,空斑形成试验测定病毒的感染性滴度。以MOI为10的病毒剂量感染HeLa细胞,PCR鉴定插入病毒的目的基因,斑点免疫印迹法鉴定目的蛋白的表达。结果pZP3α基因成功插入病毒基因组,pZP3α蛋白能在病毒感染的HeLa细胞中正常表达。病毒的颗粒性滴度约为1.3×1011VP/ml,感染性滴度约为3×109PFU/ml。结论已成功构建猪ZP3α重组腺病毒质粒pAd-pZP3α,包装并纯化了重组pZP3α腺病毒颗粒。

Construction and Purification of Recombinant Adenovirus with Porcine Zona Pellucida Gene

Objective To construct, pack and purify the recombinant adenovirus carrying porcine zona pellucida 3α(pZP3α) gene and lay a foundation of developing contraceptive vaccine. Methods The pZP3α gene was cloned into shuttle plasmid pShuttle, and the constructed recombinant plasmid pShuttle-pZP3α was co-transformed to E. coli BJ5183 with adenovirus skeleton plasmid pAd. Recombinant adenovirus pAD-pZP3α was screened and transfected to HEK293A cells in mediation of liposome, then packed and proliferated to passage 4. The recombinant virus particles were collected and purified by two rounds of cesium chloride density gradient centrifugation. The total number of virus particles (VPs)was determined by ultraviolet absorption, based on which particulate titer of virus was calculated. The infectious titer of virus was determined by plaque formation test. HeLa cells were infected with the recombinant adenovirus at a MOI of 10, based on which the target gene inserted was identified by PCR, and the expression of target protein by dot blot. Results The pZP3α gene was successfully inserted into genome of the recombinant adenovirus, and pZP3α protein was normally expressed in HeLa cells transfected with the recombinant adenovirus. The particulate and infectious titers of recombinant adenovirus reached 1. 3 × 1011 VP / ml and 3 × 109 PFU / ml respectively. Conclusion Recombinant adenovirus pAD-pZP3α was successfully constructed, packed and purified.