【摘要】：①目的:構(gòu)建沙蠶消化道上皮細(xì)胞的cDNA文庫(kù),并從中篩選、克隆和鑒定一種具有纖溶活性的蛋白編碼基因序列。②方法:分離沙蠶消化道上皮細(xì)胞,使用Trizol試劑提取總RNA,純化mRNA,用MMLV逆轉(zhuǎn)錄成cDNA,用Sfi I核酸內(nèi)切酶酶切后連接到pDNR-Lib質(zhì)粒中,構(gòu)建cDNA文庫(kù)。以SMART試劑盒中已設(shè)計(jì)好的引物行PCR擴(kuò)增篩選獲得多個(gè)克隆,將篩選成功的克隆進(jìn)行核苷酸測(cè)序,將測(cè)得的序列所編碼的相應(yīng)蛋白使用分子生物學(xué)軟件進(jìn)行功能預(yù)測(cè)和二級(jí)、三級(jí)結(jié)構(gòu)的模擬。③結(jié)果:獲得沙蠶纖溶活性蛋白的編碼序列以及插入此基因序列的重組菌。模擬結(jié)果顯示該序列編碼的蛋白與來源于哺乳動(dòng)物的絲氨酸蛋白酶相似,具有該家族保守的催化位點(diǎn)。④結(jié)論:在原核表達(dá)系統(tǒng)中成功構(gòu)建了插入此目的序列的重組菌,該基因序列編碼的蛋白有可能成為一種新型的纖溶藥物。
[Abstract]:Objective: to construct the cDNA library of digestive tract epithelial cells of silkworm, screen, clone and identify a protein coding gene sequence .2 with fibrinolytic activity: to isolate the epithelial cells of digestive tract of silkworm, silkworm. Total RNAs were extracted by Trizol reagent, mRNAs were purified, MMLV was reverse transcribed into cDNAs, then ligated into pDNR-Lib plasmids by Sfi I endonuclease. CDNA library was constructed. Several clones were obtained by PCR amplification with primers designed in the SMART kit. The cloned clones were sequenced. The corresponding proteins encoded by the detected sequences were predicted by molecular biology software. Results: the encoding sequence of fibrinolytic active protein and the recombinant bacteria inserted into the protein sequence were obtained. The simulation results show that the protein encoded by this sequence is similar to the serine protease from mammals and has the conserved catalytic site of this family. Conclusion: the recombinant bacteria inserted into the target sequence was successfully constructed in the prokaryotic expression system. The protein encoded by the gene sequence may become a new type of fibrinolytic drug.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R346

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