本文選題：雙峰駝 + 單域抗體��； 參考：《內(nèi)蒙古農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：駱駝血清中除了常規(guī)抗體IgG1,還存在兩種天然缺失輕鏈的重鏈抗體IgG2和IgG3,其具有分子量小、穩(wěn)定性強等優(yōu)點,但市場上仍缺乏商品化的駱駝重鏈抗體特異性的抗體,使其研究和應(yīng)用受到了限制,因此制備針對駱駝重鏈抗體的多抗將具有一定的應(yīng)用前景。重鏈抗體的可變區(qū)VHH是目前發(fā)現(xiàn)的、具有完整功能的最小抗體分子片段,該抗體在基礎(chǔ)研究、藥物開發(fā)等領(lǐng)域應(yīng)用前景極為廣闊。將VHH展示在噬菌體表面,構(gòu)建噬菌體抗體文庫,再通過體外篩選技術(shù)將所需性質(zhì)的多肽從含有大量變異體的集落中提取出來,為特異性抗體的制備提供了極大的便利。本研究的第一部分即結(jié)合駱駝重鏈抗體的優(yōu)勢與噬菌體展示技術(shù)的優(yōu)點,從五峰未經(jīng)免疫的健康雙峰駝外周血淋巴細胞中提取總RNA,反轉(zhuǎn)錄為cDNA,利用PCR技術(shù)擴增編碼抗體可變區(qū)的基因,通過與噬菌體載體連接,電轉(zhuǎn)化E.coli TGI感受態(tài)細胞,構(gòu)建了雙峰駝天然單域抗體文庫。經(jīng)鑒定文庫庫容為4.4×107,轉(zhuǎn)化陽性率為75%,多樣性良好。本研究第二部分,我們利用分離淋巴細胞后的雙峰駝血漿,經(jīng)過Protein G/A Resin柱子對雙峰駝三種亞型抗體進行了分離和純化,以純化的IgG2對家兔進行三次免疫制備了針對雙峰駝重鏈抗體的多克隆抗體,并對其活性進行了鑒定。間接ELISA法測定多抗效價為1：4096；間接ELISA法和Western-blot方法檢測多抗的特異性,結(jié)果顯示制備的多抗與牛IgG以及雙峰駝IgG1無交叉反應(yīng),而與IgG3抗體具有交叉反應(yīng)性。本試驗研究成功制備了雙峰駝重鏈抗體特異性多克隆抗體。
[Abstract]:In addition to the routine antibody IgG1, there are two naturally absent heavy chain antibodies, IgG2 and IgG3, which have the advantages of low molecular weight and strong stability, but there is still a lack of commercial heavy chain antibodies in the market. Therefore, the preparation of multiple antibodies against camel heavy chain antibodies will have a certain application prospect. The variable region of heavy chain antibody (VHH) is the smallest fragment of antibody with complete function, which is widely used in basic research, drug development and other fields. VHH was displayed on the phage surface to construct phage antibody library, and then the polypeptide was extracted from the colony containing a large number of variants by in vitro screening technology, which provided a great convenience for the preparation of specific antibody. The first part of this study combines the advantages of camel heavy chain antibody and phage display technology. Total RNAs were extracted from unimmunized lymphocytes of healthy bactrian camels, and then reversely transcribed into cDNAs. The genes encoding variable regions of antibodies were amplified by PCR technique, and then electrotransformed into E.coli TGI receptive cells by ligation with phage vectors. The natural single domain antibody library of Bactrian camel was constructed. The library capacity was 4.4 脳 107 and the positive rate of transformation was 750.The diversity was good. In the second part of this study, we isolated and purified three subtypes of bactrian camel by Protein G / A Resin column. The polyclonal antibody against the heavy chain antibody of Bactrian camel was prepared by three times immunization with purified IgG2 and its activity was identified. The polyclonal antibody titer of indirect ELISA assay was 1: 4096, and the specificity of indirect ELISA and Western-blot methods was determined. The results showed that the prepared polyantibodies did not cross react with bovine IgG and IgG1, but had cross reactivity with IgG3 antibody. In this study, the specific polyclonal antibody against heavy chain antibody of Bactrian camel was successfully prepared.
【學(xué)位授予單位】：內(nèi)蒙古農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S824

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