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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。
Objective: To construct human anti-human cervical scFv gene CSAs-1, and to predict its secondary structure and tertiary structure, physicochemical properties and phylogenetic tree construction. Methods: The anti-human cervical cancer CSF-1 gene CSA-1 was constructed by overlap extension PCR with hybridoma cell line CSA125 that can specifically secrete anti-human cervical cancer mAb. The structure of CSAs- Evolutionary tree construction. Results: The anti-human cervical cancer scFv gene CSAs-1 has 834bp, corresponding to a 278 amino acid polypeptide with a predicted isoelectric point of 7.215 and is a basic protein. The PHDsec secondary structure shows that CSAs-1 belongs to the α + β protein, and VH and VL Multiple protein kinase C phosphorylation sites and casein kinase II phosphorylation sites were found in the region. The tertiary structure modeling of CSAs-1 showed that VL and VH form a hydrophobic “pocket”, and Linker is free from this structure. The above results show that: CSAs-1 conforms to the structural characteristics of scFv, clearly has the spatial conformation of antigen binding sites, in theory, should have good antigen binding activity. Evolutionary analysis found that vertebrate V gene formed three evolutionary groups, CSAs-1V distribution in the evolutionary tree A group. Conclusion: The construction of a murine human anti-human cervical cancer scFv gene CSAs-1 laid the foundation for further prokaryotic and eukaryotic expression of this gene. The prediction and analysis results obtained by informatics technology are CSAs-1 expression, purification and The study of activity provides a great deal of information. It provides a basis for further analysis of the role of antibody VH, VL in antibody function, antibody modification, humanization of antibody and improvement of antibody activity.