人Ⅰ型丙氨酸氨基转移酶原核表达、表位分析及其抗体制备

发布时间：2018-06-22 01:49

本文选题：ALT_1 + B细胞抗原表位　；参考：《重庆医科大学》2011年硕士论文

【摘要】：丙氨酸氨基转移酶(Alanine aminotransferase)俗称谷丙转氨酶,它是糖异生和氨基酸代谢过程中一个至关重要的酶,主要分布在肝脏组织中。当肝组织损伤因为病毒性肝炎、非酒精性脂肪肝、肝硬化及药物因素损伤时,肝细胞中的ALT释放入血,使血清中ALT显著增高。因此,在过去的50多年间,临床上一直把血清中ALT作为反映肝功能的一个重要的生物标志物。通过对血清中ALT的检测,可以反映出肝脏功能是否损伤以及损伤的程度。近年来发现人体中ALT有3种同功酶,ALT_1、ALT_2和ALT_2-2。实验研究发现,ALT_1与ALT_2有着共同的催化活性,而ALT_2-2未发现有催化活性。ALT_1主要分布在肝脏、骨骼肌和肾脏组织中,亚细胞定位于胞浆和内质网;ALT_2主要分布在心肌细胞和骨骼肌中,而在肝脏和肾脏组织中未见表达,亚细胞定位于线粒体和内质网,在胞浆中不存在。因此,血清中ALT_1水平比总ALT能更特异的反应肝脏功能状态。目前,临床上对ALT的检测主要是通过检测总的ALT酶活性来完成的。这种方法虽然操作相对简便,结果准确,但要依赖常规的生化检测仪器,不能满足在献血现场对献血员ALT的快速筛查。我们拟建立基于免疫学诊断技术的检测方法,对血清中ALT_1的实际含量进行快速检测,以满足献血现场对血源筛查的需要,并且可以更准确、特异地反应肝脏功能。为进一步研发ALT_1免疫学体外诊断试剂奠定基础。本课题拟制备出ALT_1特异的抗体。目的: 原核表达截短的ALT_1蛋白,用它免疫新西兰大白兔后,制备出特异的ALT_1多克隆抗体;用合成的表位肽免疫BALB/c小鼠,制备特异的ALT_1单克隆抗体。为研发ALT_1免疫学体外诊断试剂奠定基础。方法: 1.利用分子克隆技术构建ALT_1截短表达载体pCold-TF-ALT_1。 2.将表达载体转化入BL21(DE3)表达宿主菌,经IPTG诱导及SDS-PAGE分析表达形式后,用镍离子亲和层析法纯化融合蛋白,再经HRV-3C蛋白酶酶切后,应用凝胶过滤法去除标签蛋白,进而得到不带标签的截短ALT_1蛋白。 3.通过B细胞抗原表位预测软件分析ALT_1的抗原表位,综合分析后,选取两段肽MC-15和CK-17交予公司合成,并分别偶联KLH以增加免疫原性。 4.用纯化的截短ALT_1蛋白免疫新西兰大白兔,制备ALT_1多克隆抗体。 5.用人工合成偶联有KLH的MC-15、CK-17表位肽分别免疫BALB/c小鼠,通过杂交瘤技术制备ALT_1单克隆抗体。结果: 1.成功构建ALT_1的截短表达质粒,通过PCR和双酶切鉴定得到与预期大小一致的基因片段,同时通过基因测序分析证实完全一致,并无移码突变。 2.通过镍离子亲和层析纯化后得到较纯的重组截短表达蛋白,融合蛋白再通过HRV-3C蛋白酶酶切后经分子筛纯化得到不带标签的目的蛋白。 3.用纯化的ALT_1截短表达蛋白免疫新西兰大白兔后,制备出了特异性好、效价高的ALT_1多克隆抗体。 4.用人工合成偶联有KLH的MC-15、CK-17表位肽免疫BALB/c小鼠,建立了多株稳定分泌ALT_1单克隆抗体的杂交瘤细胞株,通过腹水诱生法制备了腹水型ALT_1单克隆抗体,间接酶联免疫吸附试验法证明了腹水效价可达106,并可识别人血清中的ALT_1蛋白。结论: 重组表达的截短融合蛋白经镍离子亲和层析纯化后,得到的融合蛋白再通过HRV-3C蛋白酶酶切后应用分子筛得到目的蛋白,用此蛋白制备了ALT_1特异性的多克隆抗体;用合成的表位肽制备了特异性的单克隆抗体。为进一步研发ALT_1免疫学体外诊断试剂奠定基础。
[Abstract]:alanine aminotransferase ( alanine aminotransferase ) is commonly known as glutamic pyruvic transaminase , which is a crucial enzyme in the process of eogenesis and amino acid metabolism , and is mainly distributed in liver tissues .

ALT _ 1 , ALT _ 2 and ALT _ 2 - 2 were found in the liver , skeletal muscle and kidney tissues .

At present , the detection of ALT is mainly accomplished by detecting the total ALT activity . Although the method is relatively simple and accurate , it can not meet the routine biochemical detection instrument and can not meet the rapid screening of blood donors ALT in blood donation field .

In order to meet the need of blood source screening in blood donation field , we can establish the basis for further research and development of the in vitro diagnostic reagent for ALT _ 1 immunology .

Purpose :

The specific ALT _ 1 polyclonal antibody was prepared by immunizing BALB / c mice with synthetic epitope peptide , and the specific ALT _ 1 monoclonal antibody was prepared by immunizing BALB / c mice with synthetic epitope peptide .

Method :

1 . The expression vector pCold - TF - ALT _ 1 was constructed by molecular cloning .

2 . The expression vector was transformed into BL21 ( DE3 ) expression host bacteria . After IPTG induction and SDS - PAGE analysis , the fusion protein was purified by Ni ion affinity chromatography . After enzyme digestion with HRV - 3C protease , the labeled protein was removed by gel filtration , and then the label - free truncated ALT _ 1 protein was obtained .

3 . The epitope of ALT _ 1 was analyzed by B - cell antigen epitope prediction software . After comprehensive analysis , two segments of peptide MC - 15 and CK - 17 were selected to be delivered to the company for synthesis , and the immunogenicity was increased by coupling the peptide MC - 15 and CK - 17 respectively .

4 . immunizing New Zealand rabbits with purified truncated ALT _ 1 protein to prepare polyclonal antibody of ALT _ 1 .

5 . BALB / c mice were immunized with MC - 15 , CK - 17 peptide by artificial synthesis , and the monoclonal antibody of ALT _ 1 was prepared by hybridoma technique .

Results :

1 . The truncated expression plasmid of ALT _ 1 was constructed successfully , and the gene fragment consistent with the expected size was obtained by PCR and double - enzyme digestion .

2 , purifying by nickel ion affinity chromatography to obtain a pure recombinant truncated expression protein , and then carrying out molecular sieve purification on the fusion protein through the HRV - 3C protease to obtain the target protein without label .

3 . After immunizing New Zealand rabbits with purified ALT _ 1 , the polyclonal antibody with high specificity and high titer was prepared .

4 . BALB / c mice were immunized with MC - 15 , CK - 17 epitope peptide by artificial synthesis . The hybridoma cell line secreting ALT _ 1 monoclonal antibody was established . The ascites - type ALT _ 1 monoclonal antibody was prepared by the ascites - induced method . The indirect enzyme - linked immunosorbent assay demonstrated that the titer of ascites was up to 106 , and the serum ALT _ 1 protein could be identified .

Conclusion :

After the recombinant expression truncated fusion protein was purified by nickel ion affinity chromatography , the obtained fusion protein was digested with HRV - 3C protease and then the target protein was obtained by using molecular sieve . The polyclonal antibody specific for ALT _ 1 was prepared by using the protein . The specific monoclonal antibody was prepared by the synthesized epitope peptide .
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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