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【答案】应助回帖
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爱与雨下(金币+2): ！~ 2011-10-09 20:29:47
huojinlong9287(金币+100, 翻译EPI+1): 2011-10-10 09:59:00
谈不上高手，只是相互交流学习一下。注意有几处修改的地方（上传时标记被抹去了）。仅供参考，，
To construct pig’s prokaryotic expression vector pET-32a(+)-SRY and induce it to express efficiently in E. coli cell, SRY gene was amplified by (using) primer with restriction enzymes, and it was inserted into pMD19-T simple vector and transferred into the bacterium DH5α .After replication the pMD19-T-SRY recombinant plasmids were extracted, then pMD19-T-SRY and pET-32a（+）were digested by the same restriction enzymes at the same time. By this directed cloning technique, the SRY gene was inserted into pET-32a（+）expression plasmid. After PCR identifying, restriction enzyme digestion and sequencing testing, the pMD19-T-SRY recombinant plasmids were transformed into competent cell DH5α. The pMD19-T-SRY plasmids were extracted and transformed to competent cell Rosetta(DE3). pMD19-T-SRY was induced with different concentrations of isopropyl-β-D-thiogalactopyranoside (IPTG) and identified on 15% SDS-PAGE. The results showed that the fusion protein had high efficient expression in SDS-PAGE when induced by different concentrations of IPTG. |
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