抗人宫颈癌单链抗体的基因构建、空间结构和进化分析

目的:构建抗人宫颈癌单链抗体(scFv)基因CSAs-1,并进行二级结构和三级结构预测、理化性质分析及进化树构建。方法:采用重叠延伸PCR方法,以能特异性分泌抗人宫颈癌mAb的杂交瘤细胞株CSA125为原料,构建抗人宫颈癌scFv基因CSAs-1,进行测序;并通过Internet对其进行结构预测和进化树构建。结果:抗人宫颈癌scFv基因CSAs-1有834bp,对应278个氨基酸的多肽,等电点预测值7.215,为碱性蛋白质;PHDsec二级结构显示CSAs-1属于α β蛋白,VH和VL区均有多个蛋白激酶C磷酸化位点、酪蛋白激酶II磷酸化位点;CSAs-1三级结构建模显示VL和VH形成一个疏水的“口袋”,Linker游离于此结构之外。以上结果表明:CSAs-1符合scFv的结构特点,明显具有抗原结合位点的空间构象,理论上应该具有良好的抗原结合活性。进化分析发现脊椎动物V基因形成三个进化群,CSAs-1V区分布于进化树的A群中。结论:构建一个鼠源性抗人宫颈癌scFv基因CSAs-1,为该基因的进一步原核和真核表达打下了基础;应用信息学技术所获得的预测和分析结果为CSAs-1表达、纯化和活性研究提供了大量的信息;为进一步分析抗体VH、VL在抗体功能中的作用、改造抗体、实现抗体的人源化、提高抗体的活性等方面提供了一定的依据。

Construction, structure prediction and phylogenetic analysis of murine scFv gene against human cervical cancer

AIM: To construct the gene of murine single-chain Fv fragment (scFv) against human cervical cancer and to predict the physical and chemical characteristics, secondary structure, tertiary structure and the molecular evolution of the scFv using computer-assisted modeling. METHODS: The variable region gene of the heavy and light chains were amplified respectively using recombinant DNA techniques from CSA125 hybridoma cells that can secret monoclonal antibodies with high activity and specificity against human cervical cancer. These two fragments were then spliced together through a flexible linker to scFv against human cervical cancer by using splicing overlap extension (SOE) PCR. The scFv gene, named CSAs-1, was cloned into the vector pMD18-T. The physical and chemical characteristics secondary and tertiary structure and the phylogenetic tree were predicted using Internet and corresponding softwares. RESULTS: The whole scFv gene was cloned successfully and encoded 278 amino acids. Theoretically, the CSAs-1 scFv was relative molecular masses 28,800, the isoelectric point (pI) was 7.215. PHDsec indicated that CSAs-1 was alpha+beta globin. There were many protein kinase C phosphorylation sites and casein kinase II phosphorylation sites in the V(H) and V(L) domains. The computer graphic modeling indicated the linker was isolated from V(H) and V(L). The V(L), as well as the V(H), was involved in composing the hydrophobic "pocket" which was beneficial to the antigen binding. Phylogenetic tree analysis indicated that V(H) genes from various vertebrate species formed three clusters that corresponded to three V(H) groups and that V(L) sequences from each species had a level of diversity similar to that of their V(H) genes. CSAs-1 V regions belong to A group. CONCLUSION: The construction and analysis of a molecular model of scFv laid the foundation for the further research into genetic engineering antibody and the mechanism of antigen-antibody interaction.