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目的在大肠杆菌中高效表达心肌特异表达基因p93,以此作为抗原免疫产蛋母鸡,制备抗p93蛋白鸡卵黄抗体(IgY)。方法将p93基因分别插入原核表达载体pGEX-5X-1、pBV220、pET28a(+)中,选取表达量最高的载体进行表达纯化,其产物免疫产蛋母鸡。结果插入pET28a(+)中N端带有His标签的p93表达水平最高,且以包涵体形式存在,从含有pET28a-p93质粒的1L培养的菌液中可获得3mg纯化的p93蛋白。免疫产蛋母鸡,末次免疫后其卵黄抗体最高滴度达到1∶64000,Westernblot结果显示该抗体具有高度特异性。结论此抗体的制备为新基因p93的检测提供了良好的工具。
Objective To express p93 protein, a gene specific for cardiac muscle, in E.coli to produce IgY antibody against laying hens. Methods The p93 gene was inserted into prokaryotic expression vector pGEX-5X-1, pBV220 and pET28a (+), respectively. The highest expression vector was selected for expression and purification. As a result, p93 was inserted into pET28a (+) with the N-terminal His-tag at the highest level and contained in the form of inclusion bodies. 3 mg of purified p93 protein was obtained from 1L of culture solution containing pET28a-p93 plasmid. Immunization of laying hens, the final immunization yolk antibody highest titer reached 1: 64000, Western blot results show that the antibody is highly specific. Conclusion The preparation of this antibody provides a good tool for the detection of the new gene p93.