论文部分内容阅读
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 the recombinant retrovirus of glycoprotein (GP) gene of rabies virus SRV9 strain and test its immune effect on mice. Methods Plasmid pVAX-G containing the GP gene of rabies virus SRV9 strain was digested by restriction endonuclease and cloned into retroviral vector to construct recombinant plasmid pLNCX-G. Lipofectamine was used to transfect packaging cell line PA317 to screen positive clones Expand training. BHK cells were infected with virus particles. The integration and transcription of GP gene in cells were detected by PCR and RT-PCR. The virus titer was determined on NIH3T3 cells. Kunming mice were immunized with the recombinant retrovirus and the serum anti-rabies neutralizing antibody titers of the mice were tested by fluorescent antibody virus neutralization assay (FAVN). Results The constructed recombinant plasmid pLNCX-G was identified by restriction enzyme digestion. The GP gene of rabies virus has been integrated into the genome of BHK cells and can be transcribed normally. The titer of virus can reach 1.2 × 104CFU / ml. Both intramuscular and intraperitoneal injection Some mice developed anti-rabies virus neutralizing antibodies with positive rates of 6.7% and 86.7%, respectively, with effective protection rates of 0 and 76.9%, respectively. Conclusion Recombinant retrovirus of GP gene of rabies virus SRV9 strain has been successfully constructed and can induce certain neutralizing antibodies in mice, which lays the foundation for further study.