科研動(dòng)態(tài)

本研究旨在構(gòu)建表達(dá)載體pET-30a-VjbR，進(jìn)而表達(dá)布魯氏菌VjbR蛋白；利用生物軟件分析該蛋白生物信息學(xué)信息，為進(jìn)一步研究該蛋白奠定基礎(chǔ)。以布魯氏菌Rev.1株基因組為模板，擴(kuò)增VjbR基因序列，將其克隆至原核表達(dá)載體pET-30a（+）中，獲得重組質(zhì)粒pET-30a-VjbR，并進(jìn)行原核表達(dá)、Western-blot檢測(cè)及該基因的生物信息學(xué)分析。結(jié)果顯示：本試驗(yàn)成功克隆并表達(dá)了VjbR基因；經(jīng)對(duì)表達(dá)產(chǎn)物進(jìn)行SDS-PAGE分析純化，發(fā)現(xiàn)本試驗(yàn)得到較純蛋白；經(jīng)Western-blot檢測(cè)，發(fā)現(xiàn)該基因表達(dá)蛋白可與布魯氏菌羊陽性血清發(fā)生特異性反應(yīng)，具有良好的反應(yīng)原性；通過生物信息學(xué)系列軟件統(tǒng)計(jì)分析，發(fā)現(xiàn)VjbR蛋白無跨膜區(qū)，無信號(hào)肽，且該蛋白共有15個(gè)抗原決定簇，在二級(jí)結(jié)構(gòu)中，α-螺旋的氨基酸有131個(gè)，占比為49.81%。布魯氏菌VjbR基因的成功表達(dá)與純化，為進(jìn)一步制備該蛋白的單克隆抗體及iELISA診斷試劑盒的研制奠定了基礎(chǔ)。

Cloning，Prokaryotic Expression and Bioinformatics Analysis of VjbR Gene of Brucella melitensis Rev.1 Strain

In order to construct the expression vector（pET-30a-VjbR）to express the VjbR protein of Brucella，and to analyze the bioinformatics information of the protein by biological software so as to lay a foundation for further relevant study. Taking the genome of Brucella melitensis Rev.1 strain as a template，VjbR gene sequence was amplified and then cloned into the prokaryotic expression vector，pET-30a（+），to obtain the recombinant plasmid，pET-30a-VjbR，then the prokaryotic expression，western-blot and bioinformatics analysis were respectively carried out. The results showed that VjbR gene was successfully cloned and expressed；the purer protein was received after SDS-PAGE analysis and purification for the expressed products；it was detected that the expressed protein could specially react with the positive sheep serum through western-blot，showing good reactivity；according to the analysis by series of bioinformatics software，it was found that VjbR protein was with no any transmembrane domain or signal peptide，it had 15 antigenic determinants. In the secondary structure，the number of α-spiral amino acids was 13，accounting for 49.81%. In conclusion，the successful expression and purification of VjbR gene of Brucella would lay a foundation for further preparation of monoclonal antibodies and development of iELISA kit.

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