摘要
目的
构建人源性噬菌体单链抗体库，以筛选人源性抗乙型肝炎病毒表面抗原(抗HBs)的基因工程抗体。
方法
(1)抽取注射过乙肝疫苗并产生抗HBs抗体的正常人外周血，分离淋巴细胞，提取细胞总RNA
(2)以Oligo(dT)_(15)为引物将mRNA反转录为cDNA
(3)根据抗体分子的第一框架区和第四框架区的保守性，设计简并引物
(4)PCR扩增重链和轻链的可变区序列(V_H和V_L)
(5)设计一对互为模板的引物，PCR扩增连接肽Linker基因
(6)由Linker片段通过重叠延伸PCR连接扩增的V_H和V_L基因，形成单链抗体(ScFv)基因
(7)将ScFv基因克隆入具有相同酶切位点的噬菌粒载体pCANTAB5E中，然后转化大肠杆菌E.Coli TG1，辅助噬菌体M13K07的拯救，收集噬菌体上清，即为噬菌体抗体库
结果
(1) 我们设计的扩增抗体重链和轻链可变区的简并引物，成功地扩增出了V_H和V_L基因片段
(2)根据我们设计的Linker基因引物，成功地扩增出大约100bp的Linker基因(包括与V_H和V_L互补序列)
山西医科大学2003而颀十毕业论文
门）重叠延伸 PCR将 V。和 V。基因连接成约 750hp的单链抗体基
因
N）构建了 pCANTABSE沼CFV载体，转化 E．Colt TGI后，在含有
氨令青霉素的平板上长出阳性菌落，并经过PCR快速鉴定插入
片段大小的正确
（5）制备了人源性噬菌体抗体库，抗体片段以融合蛋白的形式表达
于噬菌体的表面，利于筛选
结 论
本课题通过建立人源性的噬菌体单链抗体库，以期从中筛选到
抗HBs的单链抗体，由于是人源性的，避免了鼠源性单克隆抗体
所产生的人抗鼠抗体反应，且由于是小分子的单链抗体，可以与干
扰素基因相偶联，是十分有希望的抗乙型肝炎病毒基因治疗方案，
而且为满足阻断乙肝病毒（HBV）感染的母婴垂直传播，具备生产
大量人源特异性强的抗体制品的要求。总之，抗HBS单链抗体在
临床上有着广泛的应用前景。
Objective:
Construst human genetic engineering antibody library on phage to screen human single chain antibody of anti-HBs.
Methods:
1. To separate B-lymphocytes from healthy volunteers' blood, and extract total RNA from B-lymphocytes
2. To reverse transcript mRNA into cDNA with Oligo(dT) primer according to the charater of mRNA
3.To design a set of oligonucleotide primers to amplify the cDNA of immunogloblulin heavy and light chain variable domains (Vh and Vl) by PCR
4.To amplify the linker gene by PCR to link Vh and Vl
5.To link Vh and Vl genes by a 15-amino acid linker--(Gly4Ser)3 to
form a single chain Fv (ScFv)
6.To Clone the ScFv gene into phagemid vector pCANTAB5E and tranform E.Coli TGI, with the help of the phage M13K07, so antibody fragments are expressed on the surface of phage
7.To collect the human antibody library to identify and screen our interesting antibody with ELISA
Results:
1. The mRNA from B-lymphocytes was reverse transcripted into cDNA
2. We successfully amplified Vh and Vl gene fragment ,and could see the expected bands from gel.
3. We amplified the linker gene about 100bp by PCR
4. We cloned the vector pCANTAB5E/ScFv and formed many positive clones on plate with antibiotin ampicilin
5. We collected the human phage antibody library to identify and screen the phage with anti-HBs antibody fragment
Conclusion
We expect to screen ScFv of anti-HBs by constructing a human antibody library, thus can avoid human anti-mouse antibody (HAMA) reaction. Because the ScFv is not so long as complete immnogolbulin, and hasn't the constant regions, we can connect the ScFv gene and rIFN gene to express together as a promising gene therapy method against HBV infection. Moreover, the genetic engineering antibody satify the need of many patients in order to prevent the infection of HBV between mother and baby. In a word, anti-HBs ScFv antibody has clinically extensive applied prospects.
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