本文選題：SHP-1 + 克隆��； 參考：《吉林大學》2005年碩士論文

【摘要】：為了研究SHP-1在細胞信號轉導中的生物學功能,本文根據人SHP-1催化結構域(△SHP-1)基因設計,以SHP-1全酶基因為模板,通過PCR擴增其催化部位ΔSHP-1基因,并在其N末端引入Nde I限制酶位點,C末端引入Hind III酶切位點。將PCR擴增得到的ΔSHP-1基因連入用限制性內切酶EcoR V消化的克隆載體pKS中,經Nde I和Hind III雙酶切。再用Nde I和Hind III雙酶切質粒pKS-ΔSHP-1和表達載體pT7質粒,然后用T4 DNA連接酶將ΔSHP-1與表達載體pT7連接起來,經Nde I和Hind III雙酶切,應用核酸電泳鑒定其大小正確。將表達載體轉化到E. coliBL21中,獲得了高表達菌株并進行了高效表達。 然后應用離子交換方法,通過Q-Sepharose Fast Flow、SP-Sephadex兩種離子交換柱,分離純化得到目的蛋白ΔSHP-1。透析凍干后,經SDS-PAGE、HPLC分析鑒定,其純度大于95% 。對ΔSHP-1的酶性質研究表明,當底物為p-NPP時,ΔSHP-1的最適pH為5.0,最適反應溫度為36℃,最適離子強度為0;而在pH7.5,溫度為15℃,離子強度為0條件下,對酶的活性影響最小。對其酶促反應進行動力學分析,得出Km=22.0mM,Vmax=2.901μmol/min。 我們用純化后的ΔSHP-1免疫家兔,獲得的免疫血清通過PVDF固定抗原親和層析柱制備并純化得到了抗ΔSHP-1的抗體,經ECL檢測,純化前后抗體效價均可達到1:10000(V/V);當抗原量為0.01μg時,可與純化前的抗體特異性結合,當抗原量為1ng時,可與純化后的抗體特異性結合,由此可見,抗體經過純化,對抗原的靈敏度提高了10倍;全菌電泳Weston Blot檢測,抗體純度可達到90%以上,符合免疫實驗要求。純化后抗體血清經過56℃,30min加熱滅活后,加入終濃度為1/1000的疊氮鈉,分裝小瓶,于-80℃低溫保存。 以上這些工作為在細胞水平上研究△SHP-1的生理功能以及發(fā)現它在細胞信號轉導中的作用奠定基礎。
[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，

本文編號：1895110