[抗乙肝病毒中和单克隆抗体MA18/7的融合蛋白GFP-V(L)的表达及生物活性分析]
[Expression and bioactivity analysis of the fusion protein GFP-V(L) of the neutralizing monoclonal antibody MA18/7 against HBV].
作者信息
Guan Bao-quan, Zhang Jun, Luo Wen-xin, Gu Ying, Zhu Zi-heng, Xia Ning-shao
机构信息
Ministry of Education, Key Lab of Cell Biology and Tumor Cell Engineering, Xiamen University, Xiamen 361005, China.
出版信息
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2004 Jul;20(4):444-8.
AIM
To express the fusion protein of enhanced green fluorescent protein (EGFP) with the light chain variable domain of the neutralizing monoclonal antibody MA18/7 (mAb) against hepatitis B virus in E.coli, and determine its bioactivity.
METHODS
The EGFP gene was cloned into vector pTO-T7 to construct an expression vector. And then according to ORF gene, MA18/7-V(L) was inserted into the 5' terminal of EGFP gene free of terminal code TAA to construct expression vector of fusion protein. The fusion protein was expressed in E.coli and its bioactivity was detected with ELISA and relative fluorescence intensity.
RESULTS
The expression vector EGFP-V(L) was constructed. SDS-PAGE analysis showed that expressed fusion protein was mainly in the form of inclusion body. The fusion protein retained the property of EGFP and it could bind to V(H) to form Fv which had binding activity to pre-S1.
CONCLUSION
The obtained fusion protein had good bioactivity and could be applied to further studies.
目的
在大肠杆菌中表达增强型绿色荧光蛋白（EGFP）与抗乙型肝炎病毒中和单克隆抗体MA18/7（mAb）轻链可变区的融合蛋白，并测定其生物活性。
方法
将EGFP基因克隆到载体pTO-T7中构建表达载体。然后根据开放阅读框基因，将MA18/7-V(L)插入到不含终止密码子TAA的EGFP基因5'端，构建融合蛋白表达载体。融合蛋白在大肠杆菌中表达，并用ELISA和相对荧光强度检测其生物活性。
结果
构建了表达载体EGFP-V(L)。SDS-PAGE分析表明，表达的融合蛋白主要以包涵体形式存在。融合蛋白保留了EGFP的特性，能与V(H)结合形成对前S1有结合活性的Fv。
结论
获得的融合蛋白具有良好的生物活性，可应用于进一步研究。
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