ALT1可溶性表达和单抗制备及检测方法的初步建立

发布时间：2018-01-05 11:15

本文关键词：ALT1可溶性表达和单抗制备及检测方法的初步建立　出处：《沈阳药科大学》2009年硕士论文　论文类型：学位论文

【摘要】：丙氨酸氨基转移酶(Alanine aminotransferase, ALT)是一种参与人体蛋白质新陈代谢的酶,在葡萄糖和氨基酸代谢中起着关键的调节作用。临床上,血清ALT的测定主要用于肝脏疾病的早期筛查、诊断、治疗及预后的评价。本实验克隆、表达人丙氨酸氨基转移酶基因altl。通过RT-PCR从肝癌细胞中扩增丙氨酸氨基转移酶基因alt1,并将其克隆至pET-28a(+)表达载体中。将重组表达质粒pET28a-His-ALT1转化大肠杆菌BL21,经1.0mmol/L IPTG诱导,表达可溶性重组融合蛋白His-ALT1。通过硫酸铵沉淀、镍离子柱亲和层析及QHP柱层析三步纯化重组His-ALT1蛋白,并检测融合蛋白活性及稳定性,同时制备His-ALT1蛋白冻干粉。纯化后目的蛋白纯度达85%,经测定重组蛋白具有很高的丙氨酸氨基转移酶活性(200U/mg),所制备的His-ALT1冻干粉经热稳定试验表明稳定性良好。取纯化后的His-ALT1融合蛋白作为抗原免疫BALB/c小鼠,应用淋巴细胞杂交瘤技术,制备小鼠源性抗人ALT1 McAb。用间接ELISA方法筛选出阳性杂交瘤细胞株,用有限稀释法对阳性杂交瘤细胞进行亚克隆。并制备单抗腹水,以QHP柱纯化单抗。通过过碘酸钠法以HRP标记纯化后的单抗,以Dot-blot法检测各株单抗特异性,以Dot-blot;法联合Western Blot法和阻断ELISA法进行各单抗识别抗原表位的鉴定,并以间接ELISA法分析各单抗的亲和力。用柠檬酸三钠还原法制备胶体金溶液,并制备了稳定的金标记011-3抗体,与011-8单抗组合,利用双抗体夹心免疫胶体金测定法检测人血清ALT1。用杂交瘤技术制备出十四株能稳定分泌抗人ALT1单克隆抗体的杂交瘤细胞株,分别命名为011-1、011-2至011-14。十四株单抗具有良好的特异性。Dot-blot与Western Blot结果表明：011-1、011-2、011-4、011-7、011-8、011-9、011-10七株单抗识别抗原构象表位,而011-3、011-5、011-6、011-11、011-12、011-13、011-14七株单抗识别抗原线型表位。阻断ELISA法测定结果为：011-3、011-5和011-13识别相同或相近的抗原表位；而其他十一株单抗相互之间不存在阻断现象,该十一株单抗识别不同的抗原表位。间接ELISA法检测十四株单抗的亲和力大小为：011-10011-3=011-5011-11011.1011-2011-13011-12011-4011-6=011-8011-14011-9011-7。011-8单抗与金标记011-3单抗组合,利用双抗体夹心免疫胶体金法检测血清ALT1,检测结果与生化全自动分析仪测定结果存在差异。
[Abstract]:Alanine aminotransferase (alt) is an enzyme involved in human protein metabolism. The determination of serum ALT is mainly used in early screening, diagnosis, treatment and prognosis of liver diseases. The human alanine aminotransferase gene altl was cloned and amplified by RT-PCR from hepatoma cells. It was cloned into pET-28a () expression vector. The recombinant expression plasmid pET28a-His-ALT1 was transformed into Escherichia coli BL21. The soluble recombinant fusion protein His-ALT1 was induced by 1.0 mmol / L IPTG and precipitated by ammonium sulfate. The recombinant His-ALT1 protein was purified by nickel ion column affinity chromatography and QHP column chromatography, and the activity and stability of the fusion protein were detected. His-ALT1 protein freeze-dried powder was prepared at the same time. The purity of the purified protein was 85. The recombinant protein had a high activity of alanine aminotransferase (200 Umg). The thermal stability test of the His-ALT1 freeze-dried powder shows that the stability is good. The purified His-ALT1 fusion protein was used as antigen to immunize BALB/c mice with lymphocyte hybridoma technique. Mouse anti-human ALT1 McAb. positive hybridoma cell lines were screened by indirect ELISA method and subcloned by finite dilution method. The McAbs were purified by QHP column. The McAbs were labeled with HRP by sodium periodate method. The specificity of McAbs was detected by Dot-blot method and Dot-blot was used. Western Blot method and blocking ELISA method were used to identify the antigenic epitopes of each monoclonal antibody. The affinity of each monoclonal antibody was analyzed by indirect ELISA method. Colloidal gold solution was prepared by tri-sodium citrate reduction method. A stable gold-labeled 011-3 antibody was prepared and combined with 011-8 monoclonal antibody. Double antibody sandwich immunocolloid gold assay was used to detect human serum alt 1. 14 hybridoma cell lines which could secrete monoclonal antibody against human ALT1 stably were prepared by hybridoma technique. The McAbs named 011-1, 011-2 to 011-14.#number0# strains showed good specificity. Dot-blot and Western Blot showed that: 011-1. 011-2 / 011-4 / 011-7 / 011-8 / 011-9 / 011-10 and 011-3 / 011-5 / 011-6 / 011-3 / 011-5 / 011-6, respectively. 011-11 / 12 011-12 011-13011-14 7 McAbs recognized the epitopes of antigens. The result of blocking ELISA assay was: 1 011-3. 011-5 and 011-13 recognize the same or similar epitopes; The other 11 McAbs did not block each other. The eleven McAbs recognize different epitopes. The affinity of 14 McAbs detected by indirect ELISA method is as follows:. 011-10011-3 011-5011-11011.1011-2011-13011-12011-4011-6 / 011-8011-14011. Combination of -9011-7.011-8 McAb and gold-labeled 011-3 McAb. Double antibody sandwich immunocolloid gold assay was used to detect serum alt 1, and the results were different from those of biochemical automatic analyzer.
【学位授予单位】：沈阳药科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R341

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