广西HIV-1流行毒株膜蛋白基因的扩增与纯化  

Amplification and purification of amplified fragments of env genes of HIV-1 strains in Guangxi.

作　　者：梁绍伶[1] 刘伟[1] 陈杰[1] 梁富雄[1] 李荣健[1] 

机构地区：[1]广西壮族自治区卫生防疫站,南宁530021

出　　处：《广西预防医学》2000年第3期129-132,共4页Guangxi Journal of Preventive Medicine

基　　金：卫生部全国协作课题

摘　　要：目的扩增广西HIV_1感染者的HIV_1膜蛋白基因C2_V3 区核酸片段并提纯回收 ,进行核苷酸序列测定与亚型分析。方法用淋巴细胞分离液从HIV_1感染者的静脉血中分离PBMCs,然后用多组不同的内外侧引物进行Nested_PCR扩增HIV_1膜蛋白基因C2_V3 区核酸片段 ,后用琼脂糖电泳进行提纯 ,用荧光标记末端终止物循环测序试剂盒进行测序 ,反应物用自动DNA序列分析仪进行序列测定和分析。结果经琼脂糖凝胶电泳检测 ,获得26份DNA阳性产物 ,阳性率为86.67%。不同引物的阳性率相差较大 ,根据扩增结果可以推断广西HIV_1流行毒株的基因变异程度。14份HIV_1核苷酸序列测定与亚型分析结果 ,9份为B亚型 ,5份为E亚型。结论广西存在E和B亚型HIV_1毒株的流行。HIV_1 C2_V3 region of env genes from sero_positive people in Guangxi were amplified by nested_PCR. The amplified fragments were purified by agarose gel electrophoresis and sequenced to identify the subtypes of HIV_1.Methods Firstly, PBMCs were isolated from peripheral blood samples from HIV_1 infected persons by lymphocytes separation medium. Secondly, nested_PCR was used to amplify the gene of C2_V3 region of HIV_1 with different primers and agarose gel electrophoresis to purify the products. Sequencing was performed with fluorescence_labeled terminal cycling sequencing kit. Results 26 out of 30 samples are positive, with a total positive rate of 86.67%. Positive rates are different from each other for different primers, which may deduce the variation degree of HIV_1 gene. Purified genes fragments amplified by nested_PCR of C2_V3 region were used for sequencing and detection of the subtypes of HIV_1. 9 of 14 samples from blood donors were subtype B strains and the other 5 from injecting drug users subtype E. Conclusion The amplified fragments of HIV_1 env gene by nested_PCR were able to be purified and sequenced. Positive rates of amplified fragments for different primers varied each other. There is a recent epidemic of both B and E subtype HIV_1 strains in Guangxi. [

关 键 词：HIV-1 ENV基因 NESTED-PCR 扩增 纯化 

分 类 号：R512.91[医药卫生—内科学] R373.9[医药卫生—临床医学]