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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。
OBJECTIVE: To construct a prokaryotic expression vector of GALNT3-sol, and to express and purify GALNT3-sol protein in E. coli to prepare mouse anti-human GALNT3-sol polyclonal antibody. The basic characteristics of the test. METHODS: The cDNA encoding human GALNT3-sol (1755bp) was amplified by RT-PCR and cloned into prokaryotic expression vector pET15b. The recombinant plasmid was transformed into E.coli BL21 (DE3) to express human GALNT3-sol recombinant protein. BALB / c mice were immunized with the denatured, refolded and electrophoretically purified recombinant proteins to prepare polyclonal antiserum. Antibody titer and specificity were determined by ELISA and Western blot, respectively. Results: The prokaryotic expression vector pET15b / GALNT3-sol was successfully constructed. After transfection with BL21 (DE3), the recombinant protein GALNT3-sol was efficiently expressed. The titer of mouse anti-human GALNT3-sol polyclonal antiserum was 1:25600. Have good specificity. CONCLUSION: The human GALNT3-sol protein was successfully obtained and the mouse anti-human GALNT3-sol antibody with good titer and specificity was obtained, which provided a reliable material for further studying the expression of human GALNT3 in tumor tissues.