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目的 :在大肠杆菌中表达抗HBV中和抗体MA18/7轻链可变区基因 (VL)与增强型绿色荧光蛋白 (EGFP)的融合蛋白 ,并测定其生物学活性。方法 :应用基因工程技术 ,将EGFP基因克隆到载体pTO T7中构建原核表达载体。然后将MA18/7的VL基因按照读码框插入到无终止密码子TAA的EGFP基因的 5′末端 ,构建融合蛋白表达载体。通过ELISA和相对荧光强度测定在大肠杆菌中表达的融合蛋白的活性。结果 :构建了EGFP MA18/7 VL 表达载体。SDS PAGE表明 ,融合蛋白主要以包涵体的形式存在。荧光测定显示 ,融合蛋白保持了GFP的荧光性质。ELISA的结果表明 ,融合蛋白与重链可变区 (VH)片段结合成的Fv ,能特异性地识别HBVpre S1抗原。结论 :成功地获得具有良好生物学活性的融合蛋白 ,可用于进一步的研究。
Objective: To express the fusion protein of light chain variable region gene (VL) and enhanced green fluorescent protein (EGFP) of anti-HBV neutralizing antibody MA18 / 7 in Escherichia coli and determine its biological activity. Methods: The gene of EGFP was cloned into the vector pTO T7 to construct prokaryotic expression vector. Then, the VL gene of MA18 / 7 was inserted into the 5 ’end of the EGFP gene without the stop codon TAA in the reading frame to construct a fusion protein expression vector. The activity of the fusion protein expressed in E. coli was determined by ELISA and relative fluorescence intensity. Results: EGFP MA18 / 7 VL expression vector was constructed. SDS PAGE showed that the fusion protein mainly exists in the form of inclusion body. Fluorescence assays showed that the fusion protein retained the fluorescent properties of GFP. The ELISA results showed that the fusion protein fused to the heavy chain variable region (VH) fragment can specifically recognize the HBV pre S1 antigen. Conclusion: The successful fusion protein with good biological activity can be used for further research.