猪瘟病毒E2蛋白基因重组人5型腺病毒的构建及鉴定  

Construction and identification of recombinant human adenovirus type 5 with E2 glycoprotein gene of classical swine fever virus

作　　者：杨洋[1,2] 高博[2,3] 宫婷[2,4] 李业伟[1,2] 孙程龙[1,2] 张守峰[2] 刘晔[2] 扈荣良[2] 

机构地区：[1]吉林大学畜牧兽医学院,长春130062 [2]军事医学科学院军事兽医研究所,长春130122 [3]广西兽医研究所,南宁530001 [4]吉林农业大学动物科学技术学院,长春130118

出　　处：《中国生物制品学杂志》2012年第7期805-808,815,共5页Chinese Journal of Biologicals

摘　　要：目的构建猪瘟病毒(Classical swine fever virus,CSFV)E2蛋白基因重组人5型腺病毒,并进行鉴定。方法通过巢式PCR从猪瘟活疫苗中扩增CSFV E2全基因序列,克隆至穿梭质粒pacAd5 CMVK-NpA的多克隆位点处,构建重组穿梭质粒pacAd5 CMVK-NpA-E2,将其线性化后与骨架质粒pacAd5 9.2-100共转染293AD细胞,进行重组腺病毒的包装。细胞完全病变后,采用RT-PCR法检测E2基因mRNA的转录;细胞病变法检测病毒滴度;连续传代后检测病毒的稳定性;Western blot法检测E2蛋白的表达;以105.82TCID50重组腺病毒经腹腔免疫小鼠,间接ELISA法检测小鼠血清猪瘟抗体效价。结果重组穿梭质粒pacAd5 CMVK-NpA-E2经双酶切及测序证明构建正确;重组腺病毒rAd5v-MxE2转染的293AD细胞可检测到E2基因的转录和蛋白的表达;重组腺病毒的滴度为105.82TCID50/ml;第5、30代重组腺病毒均可扩增出目的基因条带,病毒滴度无明显变化;重组腺病毒免疫小鼠后,可产生CSFV抗体。结论成功构建了CSFV E2基因重组人5型腺病毒,其免疫原性良好,为进一步研制猪瘟基因工程疫苗奠定了基础。Objective To construct and identify recombinant human adenovirus type 5 with E2 glycoprotein gene of classical swine fever virus （CSFV）. Methods The full-length E2 gene sequence was amplified from live CSFV vaccine by nested PCR and cloned to the polyclonal site of shuttle plasmid pacAd5 CMV-K-NpA. The constructed recombinant shuttle plasmid pacAd5 CMVK- NpA-E2 was linearilized and co-transfected to 293AD cells with backbone plasmid pacAd59. 2-100 for pacakging. After appearance of CPE, the transcription of E2 mRNA was determined by RT-PCR. The recombinant adenovirus was determined for titer by CPE method,and for stability after subculture. The expression of E2 protein was determined by Western blot. Kunming mice were immunized i.p. with the recombinant adenovirus at a dosage of 105. s2 TCIDs0, and determined for antibody titer against CSFV in sera by indirect ELISA. Results Restriction analysis and sequencing proved that recombinant shuttle plasmid pacAd5 CMVK-NpA-E2 was constructed correctly. Both transcription of E2 gene and expression of E2 protein were proved in 293AD cells transfected with recombinant adenovirus rAd5v-MxE2, The titer of rAd5v-MxE2 was l0s. s2 TCIDs0 / ml. Target gene fragments were amplified from rAd5v-MxE2 of passages 5 and 30, while the virus titer showed no significant change. The recombinant adenovirus induced antibody against CSFV in mice. Conclusion Recombinant human adenovirus type 5 with E2 gene of CS.FV was successfully constrcuted and showed high immunogenicity, which laid a foundation of further preparation of recombinant CSFV vaccine.

关 键 词：猪瘟病毒 E2蛋白 腺病毒 免疫原性 

分 类 号：S852.651[农业科学—基础兽医学] Q782[农业科学—兽医学]