本文關鍵詞：ALT1可溶性表達和單抗制備及檢測方法的初步建立　出處：《沈陽藥科大學》2009年碩士論文　論文類型：學位論文

  更多相關文章： 丙氨酸氨基轉移酶 克隆 表達 融合蛋白 單克隆抗體

【摘要】：丙氨酸氨基轉移酶(Alanine aminotransferase, ALT)是一種參與人體蛋白質新陳代謝的酶,在葡萄糖和氨基酸代謝中起著關鍵的調節(jié)作用。臨床上,血清ALT的測定主要用于肝臟疾病的早期篩查、診斷、治療及預后的評價。 本實驗克隆、表達人丙氨酸氨基轉移酶基因altl。通過RT-PCR從肝癌細胞中擴增丙氨酸氨基轉移酶基因alt1,并將其克隆至pET-28a(+)表達載體中。將重組表達質粒pET28a-His-ALT1轉化大腸桿菌BL21,經1.0mmol/L IPTG誘導,表達可溶性重組融合蛋白His-ALT1。通過硫酸銨沉淀、鎳離子柱親和層析及QHP柱層析三步純化重組His-ALT1蛋白,并檢測融合蛋白活性及穩(wěn)定性,同時制備His-ALT1蛋白凍干粉。純化后目的蛋白純度達85%,經測定重組蛋白具有很高的丙氨酸氨基轉移酶活性(200U/mg),所制備的His-ALT1凍干粉經熱穩(wěn)定試驗表明穩(wěn)定性良好。 取純化后的His-ALT1融合蛋白作為抗原免疫BALB/c小鼠,應用淋巴細胞雜交瘤技術,制備小鼠源性抗人ALT1 McAb。用間接ELISA方法篩選出陽性雜交瘤細胞株,用有限稀釋法對陽性雜交瘤細胞進行亞克隆。并制備單抗腹水,以QHP柱純化單抗。通過過碘酸鈉法以HRP標記純化后的單抗,以Dot-blot法檢測各株單抗特異性,以Dot-blot;法聯(lián)合Western Blot法和阻斷ELISA法進行各單抗識別抗原表位的鑒定,并以間接ELISA法分析各單抗的親和力。用檸檬酸三鈉還原法制備膠體金溶液,并制備了穩(wěn)定的金標記011-3抗體,與011-8單抗組合,利用雙抗體夾心免疫膠體金測定法檢測人血清ALT1。用雜交瘤技術制備出十四株能穩(wěn)定分泌抗人ALT1單克隆抗體的雜交瘤細胞株,分別命名為011-1、011-2至011-14。十四株單抗具有良好的特異性。Dot-blot與Western Blot結果表明：011-1、011-2、011-4、011-7、011-8、011-9、011-10七株單抗識別抗原構象表位,而011-3、011-5、011-6、011-11、011-12、011-13、011-14七株單抗識別抗原線型表位。阻斷ELISA法測定結果為：011-3、011-5和011-13識別相同或相近的抗原表位；而其他十一株單抗相互之間不存在阻斷現(xiàn)象,該十一株單抗識別不同的抗原表位。間接ELISA法檢測十四株單抗的親和力大小為：011-10011-3=011-5011-11011.1011-2011-13011-12011-4011-6=011-8011-14011-9011-7。011-8單抗與金標記011-3單抗組合,利用雙抗體夾心免疫膠體金法檢測血清ALT1,檢測結果與生化全自動分析儀測定結果存在差異。
[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.
【學位授予單位】：沈陽藥科大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R341

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