人GALNT3-sol蛋白的原核表达和抗体制备

Prokaryotic expression and antibody preparation of human GALNT3-sol protein

目的:构建人乙酰半乳糖胺转移酶3可溶性区域(GALNT3-sol)的原核表达载体,在大肠杆菌中表达并纯化GALNT3-sol蛋白,制备小鼠抗人GALNT3-sol多克隆抗体,并对抗体的基本特性进行了检测。方法:RT-PCR扩增编码人GALNT3-sol的cDNA片段(1755bp),将其克隆至原核表达载体pET15b中,转化大肠杆菌BL21(DE3)后,诱导表达人GALNT3-sol重组蛋白。将变性、复性和电泳纯化后的重组蛋白免疫BALB/c小鼠,制备多克隆抗血清。分别采用ELISA和Western blot检测抗体的效价和特异性。结果:成功构建了pET15b/GALNT3-sol原核表达载体,转化BL21(DE3)后可高效表达重组蛋白GALNT3-sol,获得的小鼠抗人GALNT3-sol多克隆抗血清效价达1∶25600,并有良好的特异性。结论:成功获得人GALNT3-sol蛋白,得到了效价和特异性良好的小鼠抗人GALNT3-sol抗体,为进一步研究人GALNT3在肿瘤组织中的表达提供了可靠的材料。

AIM： In order to detect the expression of GALNT3 in various tumor tissues, the prokaryotic expression vector of human GALNT3-sol （a truncation of GALNT3 being deleted of the hydrophobic trans-membrane domain） was constructed, and then the recombinant GALNT3-sol protein was expressed and purified from E.coli, followed by the preparation of polyclonal antibody against GALNT3-sol and characterization of its properties. METHODS： The human cDNA of GALNT3-sol （1 755 bp）was amplified from MKN45 cell line and cloned into expression vector pET5b/GALNT3-sol, then transformed into E.coli BL21（DE3）, in which the GALNT3-sol protein was induced by IPTG and then purified by Electrophoresis.Mice were immunized with the purified protein and the anti-serum was collected at different time intervals.Properties of the anti-serum were further detected by ELISA and Western blot. RESULTS： The prokaryotic expression vector of pET15b/GALNT3-sol was constructed successfully.Human GALNT3-sol protein was expressed in E.coli after IPTG induction. The titer of the obtained anti-serum reached 1∶25 600, and its specificity was proved by Western blot. CONCLUSION： Human GALNT3-sol protein can be successfully expressed in E.coli, and the specific anti-human GALNT3-sol antibody can be obtained by immunization of mice, which makes it possible to further investigate the role of GALNT3 in the progression of various tumors.