本文選題：SHP-1 + 克隆　； 參考：《吉林大學(xué)》2005年碩士論文

【摘要】：為了研究SHP-1在細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)中的生物學(xué)功能,本文根據(jù)人SHP-1催化結(jié)構(gòu)域(△SHP-1)基因設(shè)計(jì),以SHP-1全酶基因?yàn)槟０?通過(guò)PCR擴(kuò)增其催化部位ΔSHP-1基因,并在其N(xiāo)末端引入Nde I限制酶位點(diǎn),C末端引入Hind III酶切位點(diǎn)。將PCR擴(kuò)增得到的ΔSHP-1基因連入用限制性內(nèi)切酶EcoR V消化的克隆載體pKS中,經(jīng)Nde I和Hind III雙酶切。再用Nde I和Hind III雙酶切質(zhì)粒pKS-ΔSHP-1和表達(dá)載體pT7質(zhì)粒,然后用T4 DNA連接酶將ΔSHP-1與表達(dá)載體pT7連接起來(lái),經(jīng)Nde I和Hind III雙酶切,應(yīng)用核酸電泳鑒定其大小正確。將表達(dá)載體轉(zhuǎn)化到E. coliBL21中,獲得了高表達(dá)菌株并進(jìn)行了高效表達(dá)。 然后應(yīng)用離子交換方法,通過(guò)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(jīng)SDS-PAGE、HPLC分析鑒定,其純度大于95% 。對(duì)ΔSHP-1的酶性質(zhì)研究表明,當(dāng)?shù)孜餅閜-NPP時(shí),ΔSHP-1的最適pH為5.0,最適反應(yīng)溫度為36℃,最適離子強(qiáng)度為0;而在pH7.5,溫度為15℃,離子強(qiáng)度為0條件下,對(duì)酶的活性影響最小。對(duì)其酶促反應(yīng)進(jìn)行動(dòng)力學(xué)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(guò)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(jīng)ECL檢測(cè),純化前后抗體效價(jià)均可達(dá)到1:10000(V/V);當(dāng)抗原量為0.01μg時(shí),可與純化前的抗體特異性結(jié)合,當(dāng)抗原量為1ng時(shí),可與純化后的抗體特異性結(jié)合,由此可見(jiàn),抗體經(jīng)過(guò)純化,對(duì)抗原的靈敏度提高了10倍;全菌電泳W(wǎng)eston Blot檢測(cè),抗體純度可達(dá)到90%以上,符合免疫實(shí)驗(yàn)要求。純化后抗體血清經(jīng)過(guò)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(xì)胞水平上研究△SHP-1的生理功能以及發(fā)現(xiàn)它在細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)中的作用奠定基礎(chǔ)。
[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(hào)：1895110