狂犬病病毒糖蛋白基因重组逆转录病毒的构建及其免疫效果  

Construction and Immune Effect of Recombinant Retrovirus Containing Glycoprotein Gene of Rabies Virus

作　　者：赵娜[1,2] 张守峰[2] 胡永浩[1] 包世俊[1] 王颖[2] 张菲[2] 刘晔[2] 扈荣良[2] 

机构地区：[1]甘肃农业大学动物医学院,兰州730070 [2]军事医学科学院军事兽医研究所,长春130062

出　　处：《中国生物制品学杂志》2010年第4期337-340,共4页Chinese Journal of Biologicals

基　　金：国家高技术研究发展计划(863)(2006AA10A204)

摘　　要：目的构建狂犬病病毒SRV9株糖蛋白(GP)基因的重组逆转录病毒,并检测其对小鼠的免疫效果。方法酶切含狂犬病病毒SRV9株GP基因的质粒pVAX-G,将其克隆至逆转录病毒载体,构建重组质粒pLNCX-G,以脂质体介导转染包装细胞系PA317,筛选阳性细胞克隆并扩大培养。将病毒颗粒感染BHK细胞,采用PCR和RT-PCR法检测GP基因在细胞中的整合及转录,在NIH3T3细胞上测定病毒滴度。以重组逆转录病毒免疫昆明小鼠,通过荧光抗体病毒中和试验(FAVN)法检测小鼠血清抗狂犬病病毒中和抗体效价。结果所构建的重组质粒pLNCX-G经酶切鉴定正确;狂犬病病毒GP基因已整合至BHK细胞基因组中并能正常转录;病毒滴度最高可达1.2×104CFU/ml;肌肉和腹腔注射均可使部分小鼠产生抗狂犬病病毒中和抗体,其阳转率分别为6.7%和86.7%,有效保护率分别为0和76.9%。结论已成功构建了狂犬病病毒SRV9株GP基因的重组逆转录病毒,并可诱导小鼠产生一定的中和抗体,为进一步研究奠定了基础。Objective To construct a recombinant retrovirus containing the glycoprotein（GP）gene of rabies virus and determine its immune effect in mice. Methods Plasmid pVAX-G containing the GP gene of rabies virus SRV9 strain was digested with restriction endonuclease, and the obtained gene fragment was cloned into retrovirus vector. The constructed recombinant plasmid pLNCX-G was transfected to packaging cell line PA317 in mediation of liposome, and positive cell clones were screened for cloning and amplification. BHK cells were infected with virus particles, in which the integration and transcription of GP gene were determined by PCR and RT-PCR. The titer of retrovirus was determined with NIH3T3 cells. KM mice were immunized with the constructed recombinant retrovirus, and determined for serum neutralizing antibody titer against rabies virus by fluorescent antibody virus neutralization（FAVN）test. Results Restriction analysis proved that recombinant plasmid pLNCX-G was constructed correctly. The GP gene of rabies virus was integrated to the genome of BHK cells and transcribed normally. The virus titer reached 1. 2 × 104 CFU / ml at the most. The positive rates of neutralizing antibody against rabies virus induced in mice by intramuscular and intraperitoneal injections were 6. 7% and 86. 7%, while the effective protection rates were 0 and 76. 9% respectively. Conclusion The recombinant retrovirus containing the GP gene of rabies virus SRV9 strain was successfully constructed, and a certain neutralizing antibody titers were induced in mice, which laid a foundation of further study.

关 键 词：狂犬病病毒 糖蛋白 逆转录病毒 免疫效果 

分 类 号：R373.9[医药卫生—病原生物学] Q784[医药卫生—基础医学]