【作者】 王鹏；

【导师】 何於娟； 胥文春；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2011， 硕士

【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,ALT₁、ALT₂和ALT₂-2。实验研究发现,ALT₁与ALT₂有着共同的催化活性,而ALT₂-2未发现有催化活性。ALT₁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;ALT₂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ALT₁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ALT₁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ALT₁免疫学体外诊断试剂奠定基础。本课题拟制备出ALT₁特异的抗体。目的:原核表达截短的ALT₁蛋白,用它免疫新西兰大白兔后,制备出特异的ALT₁多克隆抗体;用合成的表位肽免疫BALB/c小鼠,制备特异的ALT₁单克隆抗体。为研发ALT₁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ALT₁截短表达载体pCold-TF-ALT₁。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ALT₁蛋白。3.通过B细胞抗原表位预测软件分析ALT₁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ALT₁蛋白免疫新西兰大白兔,制备ALT₁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ALT₁单克隆抗体。结果:1.成功构建ALT₁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ALT₁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ALT₁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ALT₁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ALT₁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ALT₁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ALT₁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ALT₁免疫学体外诊断试剂奠定基础。更多还原

【Abstract】 Alanine aminotransferase（ALT）,also named glutamate pyruvate transaminase, is a key enzyme for gluconeogenesis and amino acid metabolism . ALT is mainly distributed in tissue of the liver. An elevated serum ALT activity is regarded as evidence of liver damage, including hepatitis、nonalcoholic steatohepatosis、fatty liver、cirrhosis and drug hepatoxicity. Analysis of serum ALT activity levels is routinely used for detecting liver injury, a biomarker that has been in use for more than 50 years.The serum ALT is mainly detected by the method of it’s activity in the practice of medicine. Although the method for enzymic activity detection is relatively simple and accurate during operation, but to rely on conventional biochemical testing equipment, can not meet the need for rapid detection of ALT on-site the source of blood donation. And the activity assay just reflects the total serum ALT activity, rather than the actual quality of ALT, test results will reflect more exactly to the liver state.Three forms of ALT have been identified, ALT₁、ALT₂andALT₂-2. Experimental study found that, ALT₁ and ALT₂ share a common catalytic activity, while ALT₂-2 was not found to have catalytic activity. ALT₁ was mainly in the liver, skeletal muscle and kidney tissue, subcellular localization in cytoplasm and endoplasmic reticulum; while ALT₂ was mainly in cardiac cells and skeletal muscle, no expression in liver and kidney tissue, subcellular in the mitochondria and endoplasmic reticulum, not exist in the cytoplasm.Therefore, we intend to establish a diagnosis method based on immunological detection techniques, to detect the actual content of serum ALT₁ and meet the need for blood screening of blood donation on the spot. In order to establish in vitro diagnostic method for ALT₁, we project to prepare the specific antibody of ALT₁.Objective:After acquireing the truncated ALT₁ protein by prokaryotic expression system , we immunized New Zealand white rabbits for the preparation of the specific ALT₁ polyclonal antibody.Simultaneously, we immuned BALB / c mices with the synthetic peptides,and prepared the specific ALT₁ monoclonal antibody. Made the foundation to the development of in vitro diagnostic test of ALT₁.Methods:1. We constructed expression vector of the truncated ALT₁ by the technique of molecular cloning using the pCold-TF vector.2. Expression vector with ALT₁ were transformated into E.coil BL21（DE3）,then the recombinant proteins were induced by IPTG ,after beening analyzed the express pattern by SDS-PAGE, proteins were purified by Ni2+ affinity chromatography,then cut the TF tag-protein by HRV-3C protease,in the end ,we acquired the truncated ALT₁ protein by gel filtration.3. Considering results coming from different B cell epitope prediction softwares,two epitopes-MC15 and CK17, were got to prepare the ALT₁ monoclonal antibody.We Submitted the sequences to the company to synthesis peptides and coupled with the protein carrier KLH to increase the immunogenicity.4. ALT₁ polyclonal antibody was prepared after the rabbits being immunied with purified truncated protein.5. ALT₁ monoclonal antibody was prepared after BALB/c mices were immunized with the coupled peptides -MC15 and CK17.Results:1.Truncated ALT₁ expression plasmid was constructed successfully after the identification by PCR and restriction enzyme digestion,which appeared a gene fragment consistent with the expected size. Also confirmed the same by gene sequence analysis, without any frameshift mutations.2.Pure recombinant protein was acquire after being purified by Ni2+ affinity chromatography.And truncated ALT₁ protein was purified by gel filtration after being digested by HRV-3C prolease. 3. High specificity and titer ALT₁ polyclonal antibody was acquired after rabbits being immunized with purified truncated protein.4. Hybridoma cells were acquired after BALB/c mices being immunized with synthesis peptides. And ALT₁ monoclonal antibody was acquired after mices being injected with hybridoma cells though abdominal cavity.Also the McAb titer was detected up to 106 by ELISA. Certainly,the McAb could recognize the native ALT₁ in the serum.Conclusions:Pure truncated ALT₁ protein was acquired after being purified by Ni2+ affinity chromatography and gel filtration. High specificity and titer ALT₁ polyclonal antibody and monoclonal antibody were acquired after rabbits or BALB/c mices being immunized with purified truncated protein or coupled peptides,which made a foundation to the development of a rapid in vitro diagnostic test strip of ALT₁.更多还原