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  本文關(guān)鍵詞： 單克隆抗體 谷胱甘肽S-轉(zhuǎn)移酶 親和層析 原核表達(dá)　出處：《華中農(nóng)業(yè)大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

【摘要】： 在基因工程中,蛋白質(zhì)的表達(dá)經(jīng)常用到各種融合表達(dá)系統(tǒng)。GST基因融合表達(dá)體系具有蛋白表達(dá)產(chǎn)率高、表達(dá)產(chǎn)物純化方便以及利于抗體制備等優(yōu)點,得到越來越廣泛的應(yīng)用。用GST融合表達(dá)系統(tǒng)表達(dá)外源基因時,對融合表達(dá)產(chǎn)物的檢測和純化非常重要。目前GST基因融合表達(dá)系統(tǒng)表達(dá)的融合蛋白均使用市售的特定的親和層析系統(tǒng)純化如Glutathione Sepharose-4B親和層析柱,Glutathione親和柱存在價格昂貴、使用壽命有限、不適合大規(guī)模應(yīng)用等不足。為此,本試驗擬制備抗GST標(biāo)簽蛋白的特異性單克隆抗體,為利用單抗建立親和層析、免疫磁珠等表達(dá)產(chǎn)物大規(guī)模提純系統(tǒng)的研制做好前期工作,研究結(jié)果如下。 1.抗GST單克隆抗體的研制 用含pGEX質(zhì)粒的表達(dá)菌BL21表達(dá),并經(jīng)Glutathione Sepharose-4B親和層析柱純化得到高純度的GST標(biāo)簽蛋白,用純化的GST蛋白免疫Balb/c小鼠,制備免疫脾細(xì)胞,通過常規(guī)的細(xì)胞融合技術(shù)與SP2/0骨髓瘤細(xì)胞融合;用相同的純化的GST蛋白進(jìn)行篩選,所得的陽性雜交瘤細(xì)胞株,采用有限稀釋法進(jìn)行了克隆,建立了2株能持續(xù)分泌抗GST蛋白的McAb的雜交瘤細(xì)胞株3E和10G。 2.GST單克隆抗體的特性鑒定 3E和10G株單抗腹水與GST蛋白反應(yīng)的間接ELISA效價均在1×10~6以上,鑒定其亞類類型均為IgG1,染色體數(shù)目為80～94條。10G株細(xì)胞腹水與含GST標(biāo)簽的融合蛋白(AIV-NS1、IBDV-VP2、IBV-NP)的反應(yīng)性都很低,而3E株細(xì)胞腹水與三種融合蛋白反應(yīng)的間接ELISA效價均達(dá)到2×10~5以上。 3.抗GST單克隆抗體的初步應(yīng)用 用辛酸-硫酸銨法純化出3E株細(xì)胞的腹水單抗,測得純化后單抗對GST蛋白和IBDV-VP2蛋白的效價分別為1.28×10~5和6.4×10~4。而且3E株細(xì)胞單抗對GST蛋白和IBDV-VP2蛋白的相對親和力差別不大。3E株細(xì)胞單抗可用于GST融合蛋白的檢測,以及后續(xù)的利用單抗作為配基來純化GST融合蛋白。
[Abstract]:In gene engineering, various fusion expression systems. GST gene fusion expression system has the advantages of high protein expression yield, easy purification of expression products and easy preparation of antibodies. When using GST fusion expression system to express foreign gene, It is very important to detect and purify the fusion expression products. At present, the fusion proteins expressed by the fusion expression system of GST gene are all purified by specific affinity chromatography system sold on the market, such as Glutathione Sepharose-4B affinity chromatography column Glutathione affinity column. Because of its limited service life, it is not suitable for large-scale application and so on. Therefore, we intend to prepare specific monoclonal antibodies against GST tagged proteins to establish affinity chromatography by using McAbs. The large scale purification system of immunomagnetic beads and other expression products has been developed and the results are as follows. 1. Development of monoclonal antibody against GST. High purity GST tag protein was obtained by BL21 expression with pGEX plasmid and purified by Glutathione Sepharose-4B affinity chromatography. Balb/c mice were immunized with purified GST protein to prepare spleen cells. SP2/0 myeloma cells were fused with conventional cell fusion techniques, and the positive hybridoma cell lines were cloned by using the same purified GST protein. Two hybridoma cell lines 3e and 10G were established which could continuously secrete McAb resistant to GST protein. 2. Characterization of GST monoclonal antibody. The indirect ELISA titers of 3e and 10G McAb ascites reacted with GST protein were above 1 脳 10 ~ (-6). The subtypes were all identified as IgG1. The number of chromosomes was 800.94. The reactivity of AIV-NS1 / IBDV-VP2IBV-NPs was very low in AIV-NS1 / IBDV-VP2IBV-NPs. The indirect ELISA titers of the three fusion proteins in the ascites of 3e cell line were above 2 脳 10 ~ (5). 3. Application of monoclonal antibody against GST. The ascites monoclonal antibody of 3e cell line was purified by octanoic acid-ammonium sulfate method. The titers of purified McAbs to GST protein and IBDV-VP2 protein were 1.28 脳 10 ~ (5) and 6.4 脳 10 ~ (4) respectively. Moreover, the relative affinity of 3e cell McAb to GST protein and IBDV-VP2 protein was not different. 3e cell McAb could be used for the detection of GST fusion protein. GST fusion protein was purified by using monoclonal antibody as ligand.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R392

【引證文獻(xiàn)】

相關(guān)期刊論文 前2條

1 倪庚;沈金兒;熱米拉;劉娜;馮勁松;鄭曉冬;;抗Cry1c蛋白兔單克隆抗體的制備和鑒定[J];中國食品學(xué)報;2012年02期

2 沈金兒;倪庚;鄭曉冬;;抗β-葡萄糖苷酸酶(β-GUS)兔單克隆抗體的制備和鑒定[J];中國食品學(xué)報;2012年11期

相關(guān)博士學(xué)位論文 前1條

1 樂濤;多西環(huán)素殘留免疫學(xué)快速檢測技術(shù)研究[D];華中農(nóng)業(yè)大學(xué);2010年

相關(guān)碩士學(xué)位論文 前4條

1 劉宏;流行性造血器官壞死病毒ELISA檢測方法的初步建立[D];華中農(nóng)業(yè)大學(xué);2010年

2 喻翠翠;豬結(jié)合珠蛋白單克隆抗體的制備及初步應(yīng)用[D];華中農(nóng)業(yè)大學(xué);2010年

3 王建忠;GST抗體包被免疫磁珠納米佐劑的初步研究[D];華中農(nóng)業(yè)大學(xué);2011年

4 沈金兒;抗GNA兔單克隆抗體的制備及其夾心ELISA檢測體系的建立[D];浙江大學(xué);2013年

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本文編號：1505948