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XUMIN6891

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摘要：目的：構(gòu)建抗菌肽LNK-16基因6拷貝，12拷貝串聯(lián)體，并將目的基因串聯(lián)產(chǎn)物構(gòu)建到表達(dá)載體pET30a(+)上。方法：以實(shí)驗(yàn)室構(gòu)建好的LNK-16基因3拷貝的重組質(zhì)粒為模板設(shè)計(jì)引物，通過(guò)PCR擴(kuò)增合成2段3拷貝的LNK-16片段，分別命名為G1，G2。其中片段G1的上下游分別設(shè)有Nco I和Xho I酶切位點(diǎn)，片段G2的上下游分別設(shè)有EcoR I和Nco I酶切位點(diǎn)，分別把G1和G2雙酶切回收，然后將pET30a(+)載體用EcoR I ，Xho I雙酶切回收，最后在一個(gè)體系里將片段G1，G2，和pET30a(+)載體連接。這樣得到含6拷貝的LNK-16基因。然后再以它為模板設(shè)計(jì)引物，通過(guò)PCR擴(kuò)增合成2段6拷貝的LNK-16片段分別命名為G3，G4。其中G3的上下游分別設(shè)有EcoR I和Hind III酶切位點(diǎn)，G4的上下游分別設(shè)有Hind III和Xho I酶切位點(diǎn)，分別把G3和G4雙酶切回收，然后將G3，G4和經(jīng)過(guò)EcoR I 、Xho I雙酶切回收的pET30a(+)載體連接，這樣就得到含12拷貝的LNK-16基因。結(jié)果：PCR鑒定、雙酶切鑒定和DNA測(cè)序證明6拷貝，12拷貝基因的重組質(zhì)粒構(gòu)建成功。結(jié)論：該方法能方便高效地獲得所需要的多拷貝基因，為進(jìn)一步克隆到表達(dá)載體進(jìn)行高效表達(dá)打下基礎(chǔ)。

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1樓 2012-05-24 10:25:56

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XUMIN6891: 金幣+2, 翻譯EPI+1, 你用機(jī)器直譯的把？ 2012-05-24 17:27:36
愛(ài)與雨下: 翻譯EPI-1 2012-05-24 21:39:54

Abstract: Objective: To build the antimicrobial peptide LNK-16 gene 6 copy, 12 copies of the tandem body, and the target gene series products built into the expression vector pET30a (). The method: to build a good laboratory LNK-16 gene copies of the recombinant plasmid as a template design primers synthesized by PCR amplification of paragraph 2 of 3 copies of LNK-16 fragment, were named G1, G2. Fragment G1 of the upstream and downstream, respectively, with Nco I and Xho I sites upstream and downstream of the fragment of G2 with EcoR I and Nco I restriction sites, respectively, G1 and G2, double digestion recovery, and pET30a () vector using the EcoR I and Xho I double digestion recovery, the last in a system where the fragments G1, G2, and and pET30a () vector. This LNK-16 containing six copies of the gene. LNK-16 fragments and then using it as the template design primers synthesized by PCR amplification of paragraph 2 of 6 copies were named as G3, G4, Which the G3 upstream and downstream, respectively, with EcoR I and Hind III restriction sites, the G4 upstream and downstream, respectively, with Hind III and Xho I sites, respectively, G3 and G4 double digestion recovery, then to G3, G4, after the EcoR I, Xho I double digestion recovery of pET30a () vector, so that the LNK-16 gene containing 12 copies. Results: PCR identification of restriction enzyme digestion and DNA sequencing to prove that six copies of the 12 copies of recombinant plasmid was successfully constructed. Conclusion: This method can be easily and efficiently obtain the required multi-copy gene for efficient expression lay the foundation for further cloned into the expression vector.

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2樓2012-05-24 11:04:53