人SHP-1催化结构域的克隆表达及其多克隆抗体的制备

发布时间：2018-05-16 02:42

  本文选题：SHP-1 + 克隆　； 参考：《吉林大学》2005年硕士论文

【摘要】：为了研究SHP-1在细胞信号转导中的生物学功能,本文根据人SHP-1催化结构域(△SHP-1)基因设计,以SHP-1全酶基因为模板,通过PCR扩增其催化部位ΔSHP-1基因,并在其N末端引入Nde I限制酶位点,C末端引入Hind III酶切位点。将PCR扩增得到的ΔSHP-1基因连入用限制性内切酶EcoR V消化的克隆载体pKS中,经Nde I和Hind III双酶切。再用Nde I和Hind III双酶切质粒pKS-ΔSHP-1和表达载体pT7质粒,然后用T4 DNA连接酶将ΔSHP-1与表达载体pT7连接起来,经Nde I和Hind III双酶切,应用核酸电泳鉴定其大小正确。将表达载体转化到E. coliBL21中,获得了高表达菌株并进行了高效表达。 然后应用离子交换方法,通过Q-Sepharose Fast Flow、SP-Sephadex两种离子交换柱,分离纯化得到目的蛋白ΔSHP-1。透析冻干后,经SDS-PAGE、HPLC分析鉴定,其纯度大于95% 。对ΔSHP-1的酶性质研究表明,当底物为p-NPP时,ΔSHP-1的最适pH为5.0,最适反应温度为36℃,最适离子强度为0;而在pH7.5,温度为15℃,离子强度为0条件下,对酶的活性影响最小。对其酶促反应进行动力学分析,得出Km=22.0mM,Vmax=2.901μmol/min。 我们用纯化后的ΔSHP-1免疫家兔,获得的免疫血清通过PVDF固定抗原亲和层析柱制备并纯化得到了抗ΔSHP-1的抗体,经ECL检测,纯化前后抗体效价均可达到1:10000(V/V);当抗原量为0.01μg时,可与纯化前的抗体特异性结合,当抗原量为1ng时,可与纯化后的抗体特异性结合,由此可见,抗体经过纯化,对抗原的灵敏度提高了10倍;全菌电泳Weston Blot检测,抗体纯度可达到90%以上,符合免疫实验要求。纯化后抗体血清经过56℃,30min加热灭活后,加入终浓度为1/1000的叠氮钠,分装小瓶,于-80℃低温保存。 以上这些工作为在细胞水平上研究△SHP-1的生理功能以及发现它在细胞信号转导中的作用奠定基础。
[Abstract]:In order to study the biological function of SHP-1 in cell signal transduction, the catalytic site 螖 SHP-1 gene of human SHP-1 catalytic domain (SHP-1) was amplified by PCR using the whole enzyme of SHP-1 as template, according to the design of SHP-1 gene. Nde I restriction enzyme site and C terminal Hind III restriction site were introduced into the N terminal. The 螖 SHP-1 gene amplified by PCR was inserted into the clone vector pKS digested with restriction endonuclease EcoR V, and was digested by Nde I and Hind III. Then the plasmid pKS- 螖 SHP-1 was digested with Nde I and Hind III and the expression vector pT7 plasmid. Then 螖 SHP-1 was ligated with the expression vector pT7 by T4 DNA ligase. The plasmid was digested by Nde I and Hind III, and the size was identified by nucleic acid electrophoresis. The expression vector was transformed into E. coliBL21 and the high expression strain was obtained and highly expressed. Then the target protein 螖 SHP-1 was isolated and purified by ion exchange method with Q-Sepharose Fast flow cytometry SP-Sephadex. After dialysis, the purity was more than 95% by SDS-PAGEX HPLC analysis. The enzymatic properties of 螖 SHP-1 were studied. When the substrate was p-NPP, the optimum pH of 螖 SHP-1 was 5.0, the optimum reaction temperature was 36 鈩，

本文编号：1895110