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為了構(gòu)建豬SRY基因的原核表達(dá)載體pET-32a(+)-SRY，并通過(guò)誘導(dǎo)使其在大腸桿菌中獲得高效表達(dá)。本研究采用添加限制性內(nèi)切酶位點(diǎn)的引物特異性擴(kuò)增SRY基因，連入pMD19-T simple載體，轉(zhuǎn)化入大腸桿菌DH5α，克隆后提取pMD19-T-SRY陽(yáng)性重組質(zhì)粒，使用相同的內(nèi)切酶同時(shí)對(duì)pMD19-T-SRY質(zhì)粒和原核表達(dá)pET-32a（+）載體進(jìn)行酶切，連接后使SRY基因定向克隆到pET-32a(+)表達(dá)載體中。經(jīng)PCR、酶切和測(cè)序鑒定后，重組質(zhì)粒轉(zhuǎn)化大腸桿菌感受態(tài)DH5α，提取質(zhì)粒后再次轉(zhuǎn)化大腸桿菌Rosetta（DE3），用不同濃度的異丙基硫代半乳糖苷（IPTG）誘導(dǎo)表達(dá)，并通過(guò)15% SDS-PAGE鑒定。結(jié)果顯示，不同濃度IPTG誘導(dǎo)的SRY基因均在大腸桿菌中進(jìn)行了高效特異性融合表達(dá)。
To construct prokaryotic expression vector pET-32a(+)-SRY of pig and induced it expresses 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 for replication. The pMD19-T-SRY recombinant plasmids were extracted, then pMD19-T-SRY and pET-32a（+）were digested with same restriction enzymes. By this directed cloning technique, the SRY gene was inserted into pET-32a（+）expression plasmid. After identified by PCR, restriction enzyme digestion and sequencing, 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 detected on 15% SDS-PAGE. The results showed that the fusion protein were specifically high efficient expression in SDS-PAGE after induced by different concentrations of IPTG.

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1樓 2011-10-09 16:44:04

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xiaokaizi

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愛與雨下(金幣+2): ！~ 2011-10-09 20:29:47
huojinlong9287(金幣+100, 翻譯EPI+1): 2011-10-10 09:59:00

談不上高手，只是相互交流學(xué)習(xí)一下。注意有幾處修改的地方（上傳時(shí)標(biāo)記被抹去了）。僅供參考，，
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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2樓2011-10-09 20:19:54

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