HIV-1 GP120主要抗原性片段的制备及其在HIV诊断中的应用

目的研制HIV中国流行株RL42GP120主要抗原性片段,以便进一步开发HIV抗体检测试剂。方法克隆RL42毒株GP120蛋白C-端250个氨基酸的编码序列,克隆入大肠杆菌表达载体pBV220进行表达。通过包涵体处理和离子交换层析纯化目的蛋白。将纯化后的蛋白用狭缝电转技术转印硝酸纤维素膜,利用HIV参考品血清检测其灵敏度和特异性。结果目的蛋白的表达量占菌体总蛋白的10%左右。纯化后其纯度高于95%,HIV参考品血清检测显示出良好的灵敏度和特异性,检测8份阳性临床标本无一漏检,而7份阴性临床标本无交叉反应。结论已研制出具有良好灵敏度和特异性的HIV-1GP120主要抗原性片段。为开发HIV抗体检测试剂创造了条件。

Preparation of Major Antigen Fragment of HIV-1 GP120 and Its Application in Diagnosis of HIV

Objective To prepare the major antigen fragment of GP120 of HIV-1 virus strain RL42 epidemic in China and develop a diagnostic kit for HIV antibody.Methods The gene encoding the 250 amino acids at C-terminus of GP120 of strain RL42 was amplified and subcloned into E.coli expression vector pBV220.After themo-induction,the recombinant protein was purified by washing of inclusion body,precipitation with ammonium sulfate and ion exchange chromatography(IEC).The purified protein was prepared into antigen strips for immunoblot assay with HIV reference sera at various dilutions.Eight HIV-positive and seven HIV-negative serum specimens isolated in clinic were tested with the antigen strips.Results The expressed product contained about 10% of total somatic protein and reached a purity of more than 95% after purification.Strip immunoblot assay showed a clear reaction band of the recombinant GP120 antigen with 1∶20 000 diluted HIV-positive serum.The antigen strip prepared with the purified protein reacted specifically with eight HIV-positive serum specimens but showed no cross reaction with seven HIV-negative specimens.Conclusion The major antigen fragment of HIV-1 GP120 with good specificity and sensitivity was prepared.It laid a foundation of development of diagnostic kit for HIV antibody.