本文選題：LSm基因 + 巢式PCR��； 參考：《中國畜牧獸醫(yī)》2017年06期

【摘要】：為分析LSm1基因的特點(diǎn),預(yù)測其編碼蛋白的生物學(xué)功能,試驗(yàn)利用巢式PCR方法對Marc145細(xì)胞源LSm1基因進(jìn)行擴(kuò)增、克隆及序列測定,應(yīng)用生物信息學(xué)方法對Marc145細(xì)胞源LSm1基因進(jìn)行序列分析,并對其編碼蛋白進(jìn)行二級結(jié)構(gòu)、B細(xì)胞表位、保守結(jié)構(gòu)域、跨膜結(jié)構(gòu)域和信號肽預(yù)測。結(jié)果顯示,經(jīng)過兩輪的PCR擴(kuò)增,成功獲得402bp的Marc145細(xì)胞源LSm1基因,編碼133個氨基酸。Marc145細(xì)胞源LSm1基因核苷酸序列與人類、靈長類同源性較高,其次是海洋哺乳動物類,最后是陸地野生動物及家畜,同源性為92.8%~99.8%,而其編碼的氨基酸雖沒有此規(guī)律,但氨基酸序列同源性均較高,為97.8%~99.3%。系統(tǒng)進(jìn)化樹結(jié)果顯示,Marc145細(xì)胞源LSm1基因與人類LSm1基因親緣關(guān)系較近,處在同一分支,其次是靈長類。LSm1蛋白二級結(jié)構(gòu)中α-螺旋和無規(guī)則卷曲所占比例較大,分別為45.11%和24.06%。預(yù)測此蛋白存在4個B細(xì)胞優(yōu)勢抗原表位,具有Sm超家族保守結(jié)構(gòu)域,無跨膜區(qū)域,無信號肽區(qū)域。結(jié)果表明,LSm1蛋白在細(xì)胞內(nèi)轉(zhuǎn)錄及表達(dá)水平可能較低,細(xì)胞cDNA需通過巢式PCR擴(kuò)增兩輪才能出現(xiàn)目的條帶,本試驗(yàn)方法為LSm1基因的擴(kuò)增提供了參考。同時,LSm1生物學(xué)功能預(yù)測結(jié)果為獲得LSm1人工表達(dá)蛋白與針對LSm1蛋白的特異性抗體制備,以及在細(xì)胞水平研究LSm1的功能機(jī)制及其在病毒復(fù)制中的作用奠定基礎(chǔ)。
[Abstract]:In order to analyze the characteristics of LSm1 gene and predict the biological function of its encoded protein, the LSm1 gene derived from Marc145 cells was amplified, cloned and sequenced by nested PCR method. Marc145 cell-derived LSm1 gene was sequenced by bioinformatics, and its encoded protein was predicted by secondary structure B cell epitope, conserved domain, transmembrane domain and signal peptide. The results showed that after two rounds of PCR amplification, the Marc145 cell-derived LSm1 gene of 402bp was successfully obtained. The nucleotide sequence of the LSm1 gene encoding 133 amino acids. Marc145 cells was highly homologous to human and primates, followed by marine mammals. Finally, wild animals and domestic animals on land, the homology was 92.8% and 99.8%, while the amino acids encoded by them had no such rule, but the amino acid sequence homology was 97.8% (99.3%), and the amino acid sequence was 97.8% (99.3%). Phylogenetic tree results showed that LSm1 gene derived from Marc145 cells was closely related to human LSm1 gene and was in the same branch, followed by 偽 -helix and irregular curl in the secondary structure of primate .LSm1 protein, which were 45.11% and 24.06%, respectively. It was predicted that the protein had four B cell epitopes with Sm superfamily conserved domain, no transmembrane domain and no signal peptide region. The results showed that the transcriptional and expression level of LSm1 protein in cells may be low, and the target bands of cDNA could be obtained by nested PCR amplification. This method provides a reference for the amplification of LSm1 gene. At the same time, the prediction of the biological function of LSm1 lays a foundation for the preparation of LSm1 artificially expressed protein and the specific antibody against LSm1 protein, and for the study of the functional mechanism of LSm1 and its role in virus replication at the cellular level.
【作者單位】： 貴州大學(xué)動物科學(xué)學(xué)院;貴州省動物疫病與獸醫(yī)公共衛(wèi)生重點(diǎn)實(shí)驗(yàn)室;貴州大學(xué)大數(shù)據(jù)與信息工程學(xué)院;
【基金】：國家自然科學(xué)基金:P-body在PRRSV復(fù)制和持續(xù)感染過程中的作用機(jī)制研究(國科金計(jì)項(xiàng)[2014]44) 貴州省科學(xué)技術(shù)基金:NSP2亞類在PRRSV復(fù)制過程中的作用機(jī)制研究(黔科合J字[2015]2046號)
【分類號】：Q78;Q811.4
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本文編號：1794519