生长抑素基因工程天然及免疫鼠源单链抗体库构建研究

发布时间：2019-03-30 09:19

【摘要】： 噬菌体抗体库技术是继单克隆抗体制备技术后具有换代地位的抗体制备技术,在功能蛋白的表达、疾病诊断和治疗等领域具有广泛的应用前景。生长抑素(SS)是由下丘脑分泌的一种神经多肽,通过抑制生长激素释放而抑制动物生长和泌乳,是重要的内分泌激素。因此,建立SS免疫测定方法和免疫芯片技术对于研究动物生长和泌乳调控机制、开发促进动物生长发育和泌乳的新技术等,均具有重要意义。迄今,国内外关于小分子单链抗体的研究鲜见报道,至今无应用噬菌体展示技术筛选生长抑素单链重组抗体的报道。本研究取健康BALB/C小鼠脾脏和用偶联牛血清白蛋白的SS免疫小鼠脾脏,分别提取总RNA,扩增抗体轻链和重链可变区基因,组装成单链抗体基因。借助噬菌体展示技术,平行建立天然和免疫鼠源单链抗体库。主要结果如下: 1.人工抗原的合成及免疫:将设计合成的生长抑素利用碳化二亚胺法与BSA偶联,制备人工抗原,免疫BalB/C小鼠,其抗血清效价最高可达1:2560。 2.抗体可变区基因的获得及目的基因构建:分别取未免疫的和抗血清效价较高的小鼠脾脏提取总RNA,用RT-PCR分别扩增得到抗体重链可变区(V_H)和轻链可变区(V_L)基因,大小分别为360bp和340bp左右;纯化回收的V_H与V_L基因在接近等摩尔浓度下连接。在免疫库构建中采用含有Linker接头片段的引物,利用重叠延伸PCR法(SOE-PCR)将V_H与V_L基因片段拼接起来,再用带有限制性内切酶位点的引物扩增出单链抗体(ScFv)基因片段;在天然库构建中采用不对称PCR连接单链抗体(ScFv)基因片段,分别获得大小约750bp的目的片段。结果表明,不对称PCR连接方法的效率高于传统的组装方法。 3.ScFv噬菌体抗体库构建:ScFv基因纯化后经SfiI和NotI双酶切,与同样经过双酶切的载体pCANTAB5E连接,电穿孔法转化大肠杆菌TG1感受态细胞,提取质粒,经PCR初步鉴定证明有外源片段插入且与目的片段一致。转化菌经辅助噬菌体M13K07超感染后,收集上清,得到噬菌体抗体库。天然库的构建中,经过40次电击,获得了9.8×10~7抗体库,相对于免疫抗体库的3条片段SOE连接法,5次电击获得的9.3×10~7抗体库,后者的效率要高的多。可以看出,免疫抗体库构建的工作量远远低于天然库。该抗体库的建立,为进一步筛选和表达抗体、建立生长抑素放射免疫测定、酶免疫测定、免疫组化分析和免疫芯片等技术奠定了基础。
[Abstract]:Phage antibody library (phage antibody library) is an antibody preparation technique which has the status of substitution after the preparation of monoclonal antibodies. It has wide application prospects in the fields of functional protein expression, disease diagnosis and treatment, and so on. Somatostatin (SS) is a neuropeptide secreted by hypothalamus. Somatostatin is an important endocrine hormone which inhibits animal growth and lactation by inhibiting growth hormone release. Therefore, it is of great significance to establish SS immunoassays and immuno-chip technique to study the regulation mechanism of animal growth and lactation, and to develop new techniques to promote animal growth and lactation. Up to now, there have been few reports on the study of small scFv and no report on screening somatostatin scFv by phage display technique. In this study, the spleens of healthy BALB/C mice and the spleens of mice immunized with bovine serum albumin (SS) were used to extract the variable region genes of the light chain and heavy chain of the amplified antibodies, respectively, and to assemble the single chain antibody genes into the spleens. The phage display technique was used to construct the natural and immune mouse scFv library in parallel. The main results are as follows: 1. Synthesis and immunization of artificial antigen: the synthetic somatostatin was coupled with BSA by carbodiimide method to prepare artificial antigen and immunize BalB/C mice. The antiserum titer was up to 1? 2560. 2. Acquisition of variable region gene of antibody and construction of objective gene: total RNA, was extracted from spleen of mice with high titer of anti-serum and unimmunized mice. The heavy chain variable region (V / H) and light chain variable region (V / L) of the antibody were amplified by RT-PCR, and their sizes were about 360bp and 340bp, respectively. The purified V _ (H) and V ~ (?) L genes were linked to each other at the same molar concentration. In the construction of immune library, the primers containing Linker splice fragment were used to splice the V _ (?) H with V\ The scFv (ScFv) gene fragment was amplified by primer with restriction endonuclease site. In the construction of natural library, asymmetric PCR was used to ligate the single chain antibody (ScFv) gene fragment, and the target fragment of about 750bp was obtained respectively. The results show that the efficiency of asymmetric PCR connection method is higher than that of traditional assembly method. Construction of 3.ScFv phage antibody library: ScFv gene was purified and digested by SfiI and NotI, then ligated with vector pCANTAB5E, and transformed into E. coli TG1 competent cells by electroporation, then the plasmid was extracted. It was proved by PCR that the foreign fragment was inserted and consistent with the target fragment. After superinfection with assistant phage M13K07, phage antibody library was obtained by collecting supernatant of transformed bacteria. In the construction of natural reservoir, 9.8 脳 10 ~ 7 antibody library was obtained after 40 electric shocks. Compared with the three fragments SOE connection method of immune antibody library, the 9.3 脳 10 ~ 7 antibody library obtained by five electric shocks was much more efficient than that of immune antibody library. It can be seen that the workload of constructing immune antibody library is much lower than that of natural one. The establishment of the antibody library lays a foundation for further screening and expression of antibodies, establishment of somatostatin radioimmunoassay, enzyme immunoassay, immunohistochemical analysis and immuno-chip techniques.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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