狂犬病病毒糖蛋白和核蛋白基因的克隆及其重组蛋白在昆虫细胞中的表达  

Cloning and expression of glycoprotein and nucleoprotein of rabies virus

作　　者：苏华[1] 张鹏飞[1] 

机构地区：[1]卫生部北京生物制品研究所

出　　处：《中华微生物学和免疫学杂志》1998年第5期363-3,共1页Chinese Journal of Microbiology and Immunology

摘　　要：目的从国内狂犬病疫苗ａＧ株中克隆狂犬病病毒糖蛋白（ＧＰ）基因和核蛋白（ＮＰ）基因，应用杆状病毒－昆虫细胞表达系统使其在昆虫细胞中表达。方法从狂犬病病毒感染细胞上清液中提取病毒ＲＮＡ，应用ＲＴ－ＰＣＲ方法扩增ＧＰ基因和ＮＰ基因。扩增的基因与转移质粒连接并转化大肠杆菌，得到重组转移质粒。将其与野生杆状病毒（ＡｃＭＮＰＶ）线性ＤＮＡ共转染Ｓｆ９昆虫细胞，通过有限稀释法筛选含有ＧＰ基因和ＮＰ基因重组病毒。初步检测了重组蛋白的抗原性。结果用ＲＴ－ＰＣＲ方法扩增得到ＧＰ基因和ＮＰ基因，通过重组转移后重组病毒感染的细胞经免疫印染实验表明可分别表达ＧＰ和ＮＰ重组蛋白。重组蛋白的分子量分别为５８×１０３和５３×１０３。用重组病毒感染的细胞免疫小鼠后可诱导动物产生特异性抗体。结论可以应用杆状病毒－昆虫细胞表达系统表达狂犬病病毒ＧＰ和ＮＰ重组蛋白。Objective To stusy the cloning of the genes of glycoprotein and nucleoprotein of rabies virus and expression of the recombinant protein G and N in insect cells by using the baculovirus insect cell expression system. Methods The genes coding for GP and NP of rabies virus were obtained from viral RNA extracted from supernatant of viral culture by RT PCR, and were cloned into baculovirus transfer vectors,respectively. Sf9 cells were co transfected with recombinant vectors and wild type baculovirus linear DNA, the recombinant baculovirus containing the genes of GP and NP were isolated through several rounds of limited dilution. The antigenicity and the immunogenicity of the recombinant proteins were investigated preliminarily. Results The genes coding for GP and NP of rabies virus were obtained through RT PCR. The recombinant proteins G and N were expressed in Sf9 cells post infected with recombinant baculoviruses,which were evidenced by recognition of recombinant proteins with both Western blot and ELISA. The molecular weight of recombinant protein G and N were about 58kD and 53kD,respectively. The results of immunization assay with recombinant baculoviruses infected cell lysates indicated that the recombinant proteins expressed in insect cells had the ability to elicit specific antibodies against native rabies virus in mice. Conclusions The baculovirus insect cell expression system can be used to express the genes coding for GP and NP of rabies virus and the recombinant proteins could induce specific antibodies in mice.

关 键 词：狂犬病病毒 糖蛋白 核蛋白 昆虫细胞 基因表达 

分 类 号：R373.9[医药卫生—病原生物学] Q78[医药卫生—基础医学]