乙型肝炎病毒表位PreS1基因工程抗体的获得——天然及免疫人源单链抗体库的构建
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摘要
乙型肝炎病毒(HBV)感染是公认的防治难题。抗体性药物是乙型肝炎治疗领域的新方向之一。随着噬菌体展示技术的发展，抗体库的构建方案日趋成熟。全人源化乙型肝炎病毒基因工程抗体逐渐成为这一领域的研究热点。
抗体在保护性免疫过程中起着决定性作用。针对具体抗原表位的抗体才能满足预防感染或治疗的需要。乙型肝炎表面抗原(HBSAg)颗粒表面的PreS1抗原，是与HBV的侵袭密切相关的抗原表位。人源化高亲和力的Pres1抗体，将在抗体药物治疗HBV感染这一领域发挥作用。
本研究以抗体多样性的遗传机制为理论基础，借助噬菌体展示技术，以应用于临床治疗为目标，直接介入抗体工程的最前沿，全人源化基因工程抗体中的单链抗体。结合保护性免疫的最新进展，针对乙型肝炎病毒表位Pres1，为获得抗体性药物作了前期研究工作。
本研究以健康供血者的外周血淋巴细胞为来源，部分以偶联BSA的PreS1肽体外免疫后，分别提取总RNA，扩增抗体轻链和重链可变区基因，组装成单链抗体(scFv)基因。借助噬菌体展示技术，平行构建了天然及免疫人源单链抗体库。再以PreS1肽进行固相淘选，两个抗体库均获得了PreS1单链抗体。
本研究首次获得了两个乙型肝炎病毒表位Pres1单链抗体ZG1、ZG2的基因。同时对比了两个抗体库淘选结果：免疫库的多克隆ELISA、单克隆ELISA结果均优于天然库；每轮淘选的结果表明免
大连理工大学博士学位论文
疫库次级库的容量超过天然库4－50倍。所以，在体外免疫阶段淋巴
细胞的抗体基因趋向抗原进行了重排，目的抗体基因丰度得到提高。
竞争ELISA 也证实，来自免疫库的单链抗体ZGI 亲和力为
10’乙 10－‘M，高于来自天然库的 ZGZ，亲和力 10‘7一”’M。说明体外
免疫是成功的。这一结果表明取材于体外免疫的淋巴细胞构建免疫
抗体库是获得高亲和力抗体的捷径。
为了优化抗体基因的多样性，本研究完善了抗体库的构建方法。
在单链抗体组装过程中，我们首次采用了通过不对称PCR扩增单链
DNA，再俩俩连接的方法。结果表明，这种方法的效率高于传统的
组装方法。
总之，本研究首次获得了高亲和力的 PreS 人源单链抗体及其
基因，为乙型肝炎的人源化抗体治疗做了前期研究。该单键抗体基
因已经申请了国家发明专利，发明名称：抗乙型肝炎病毒 PreS 抗
原的人源单链抗体。申请号为 01 127973．7。
As known to all, therapy of Hepatitis B is something of difficulties. Until now only immune enhancers such as interferon and thymus peptide are clinically utilized. But their actions are not certain. Today, study of antibody medicine against HBV is newly in this field. Accompanied the development of phage display technology, the construction of antibody library is becoming matured. And the research of completely humanized antibody against HBV is focused on.
Antibodies play a crucial role in protective immune, among which only that against epitope can satisfy prevention or therapy. PreS 1 is one of the important epitope of HBsAg, which has been pointed out to have relationship with infection of HBV. Humanized antibody against PreSl with high affinity is a new way for blocking or cleaning HBV.
This study interested in the most advanced field: completely humanized genetic antibody. Based on diversity of antibody and aimed at clinical therapy, it studied genetic antibody against PreSl according to recent advance of protective immunity. This study was taken as a basic research for antibody therapy.
Two scFv libraries were constructed paralleled via phage display technique. PBLs were from healthy donors and divided into two parts. One was in vitro immunized by PreSl peptide, the other was natural. Then the two were used to amplify antibody genes for the naive and immune library respectively.
High affinity scFvs against PreSl were obtained from both two libraries after panning against PreSl peptide, so were the genes encoding them. Comparison of the two libraries showed, monoclonal ELISA and polyclonal ELISA of immune library were better than that of naive one. The size of immune library during every round of panning was 4-50 folds bigger than that of the naive one. It is reasonable
to consider that genes of lymphocytes were rearranged to the antigen during in vitro immunization. Competed ELISA confirmed further that the higher affinity scFv was from immune lib. All of above testify it is the expeditious way to get higher affinity scFv from in vitro immunized antibody library.
This study obtained the high affinity human scFv against PreS 1 for the first time. It is the foundation of therapy of Hepatitis B. The patent of the gene encoding this scFv is processing, number of which is 01127973.7.
抗体在保护性免疫过程中起着决定性作用。针对具体抗原表位的抗体才能满足预防感染或治疗的需要。乙型肝炎表面抗原(HBSAg)颗粒表面的PreS1抗原，是与HBV的侵袭密切相关的抗原表位。人源化高亲和力的Pres1抗体，将在抗体药物治疗HBV感染这一领域发挥作用。
本研究以抗体多样性的遗传机制为理论基础，借助噬菌体展示技术，以应用于临床治疗为目标，直接介入抗体工程的最前沿，全人源化基因工程抗体中的单链抗体。结合保护性免疫的最新进展，针对乙型肝炎病毒表位Pres1，为获得抗体性药物作了前期研究工作。
本研究以健康供血者的外周血淋巴细胞为来源，部分以偶联BSA的PreS1肽体外免疫后，分别提取总RNA，扩增抗体轻链和重链可变区基因，组装成单链抗体(scFv)基因。借助噬菌体展示技术，平行构建了天然及免疫人源单链抗体库。再以PreS1肽进行固相淘选，两个抗体库均获得了PreS1单链抗体。
本研究首次获得了两个乙型肝炎病毒表位Pres1单链抗体ZG1、ZG2的基因。同时对比了两个抗体库淘选结果：免疫库的多克隆ELISA、单克隆ELISA结果均优于天然库；每轮淘选的结果表明免
大连理工大学博士学位论文
疫库次级库的容量超过天然库4－50倍。所以，在体外免疫阶段淋巴
细胞的抗体基因趋向抗原进行了重排，目的抗体基因丰度得到提高。
竞争ELISA 也证实，来自免疫库的单链抗体ZGI 亲和力为
10’乙 10－‘M，高于来自天然库的 ZGZ，亲和力 10‘7一”’M。说明体外
免疫是成功的。这一结果表明取材于体外免疫的淋巴细胞构建免疫
抗体库是获得高亲和力抗体的捷径。
为了优化抗体基因的多样性，本研究完善了抗体库的构建方法。
在单链抗体组装过程中，我们首次采用了通过不对称PCR扩增单链
DNA，再俩俩连接的方法。结果表明，这种方法的效率高于传统的
组装方法。
总之，本研究首次获得了高亲和力的 PreS 人源单链抗体及其
基因，为乙型肝炎的人源化抗体治疗做了前期研究。该单键抗体基
因已经申请了国家发明专利，发明名称：抗乙型肝炎病毒 PreS 抗
原的人源单链抗体。申请号为 01 127973．7。
As known to all, therapy of Hepatitis B is something of difficulties. Until now only immune enhancers such as interferon and thymus peptide are clinically utilized. But their actions are not certain. Today, study of antibody medicine against HBV is newly in this field. Accompanied the development of phage display technology, the construction of antibody library is becoming matured. And the research of completely humanized antibody against HBV is focused on.
Antibodies play a crucial role in protective immune, among which only that against epitope can satisfy prevention or therapy. PreS 1 is one of the important epitope of HBsAg, which has been pointed out to have relationship with infection of HBV. Humanized antibody against PreSl with high affinity is a new way for blocking or cleaning HBV.
This study interested in the most advanced field: completely humanized genetic antibody. Based on diversity of antibody and aimed at clinical therapy, it studied genetic antibody against PreSl according to recent advance of protective immunity. This study was taken as a basic research for antibody therapy.
Two scFv libraries were constructed paralleled via phage display technique. PBLs were from healthy donors and divided into two parts. One was in vitro immunized by PreSl peptide, the other was natural. Then the two were used to amplify antibody genes for the naive and immune library respectively.
High affinity scFvs against PreSl were obtained from both two libraries after panning against PreSl peptide, so were the genes encoding them. Comparison of the two libraries showed, monoclonal ELISA and polyclonal ELISA of immune library were better than that of naive one. The size of immune library during every round of panning was 4-50 folds bigger than that of the naive one. It is reasonable
to consider that genes of lymphocytes were rearranged to the antigen during in vitro immunization. Competed ELISA confirmed further that the higher affinity scFv was from immune lib. All of above testify it is the expeditious way to get higher affinity scFv from in vitro immunized antibody library.
This study obtained the high affinity human scFv against PreS 1 for the first time. It is the foundation of therapy of Hepatitis B. The patent of the gene encoding this scFv is processing, number of which is 01127973.7.
引文
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