副结核分枝杆菌胞外GDSL脂肪酶MAP_2739的酶学特性研究

Enzymatic characteristics of extracellular GDSL lipase MAP_2739 from Mycobacterium avium subsp.Paratuberculosis

本实验室前期研究发现了未见报道的副结核分枝杆菌(MAP)GDSL脂肪酶家族蛋白MAP_2739。为进一步分析MAP_2739的酶学特性,本实验利用生物信息学网站和软件对MAP_2739保守结构域、二级结构、3D同源建模等分析,进一步确认其属于GDSL脂肪酶家族蛋白。以MAP参考株基因组DNA为模板,PCR扩增获得map_2739目的基因约891 bp,克隆于表达载体pET-22b中,构建重组质粒pET-22b-map_2739,转化大肠杆菌感受态细胞,经IPTG诱导表达、蛋白复性、Ni-TNA柱亲和层析纯化后,利用SDS-PAGE分析目的蛋白的表达及纯化效果,并通过western blot鉴定。结果显示,获得了以包涵体形式表达的经复性的纯化重组MAP_2739蛋白(rMAP_2739)。利用该蛋白制备兔多克隆抗体,通过ELISA方法检测,多抗血清效价为1∶51200。经差速离心法分离MAP参考株组分,利用该多克隆抗体为一抗,通过western blot检测,结果显示在细胞壁、细胞膜和细胞浆均出现35 ku的特异性条带,表明MAP_2739为胞外蛋白。以对硝基苯基酯(p-NPs)为底物进行酶活测定,结果显示MAP_2739蛋白为脂肪酶,可水解短链和长链脂肪酸,其最适酶反应pH值为9.0,且rMAP_2739蛋白经巴氏消毒法预处理后,仍具有脂肪酶活性。以重组质粒pET-22b-map_2739为模板,利用各定点突变引物,经PCR扩增各突变的map_2739基因,并测序后转化大肠杆菌感受态细胞,通过western blot鉴定蛋白表达及纯化情况,并进行了酶活测定分析,结果显示获得各定点突变纯化蛋白,确定S46和D204为MAP_2739的酶活性位点。综上所述,本研究首次证明MAP_2739是MAP胞外GDSL脂肪酶,该结果为进一步研究其在MAP致病中的作用机制奠定了基础。

In the early stage of this study,an unreported GDSL lipase family protein of Mycobacterium avium subsp.paratuberculosis(MAP),MAP_2739 was discovered.To study the enzymatic properties of MAP_2739,bioinformatics websites and software were used to analyze the conserved domain,secondary structure and 3D homology modeling of MAP_2739,and further confirmed that it belonged to the GDSL lipase family.The target gene of MAP_2739 was obtained by PCR amplification of about 891bp using the genomic DNA of MAP reference strain as a template,ligated to the expression vector pET-22b,verified by sequencing,and successfully constructed a recombinant expression strain of E.coli,which was purified by IPTG-induced expression,protein refolding,and affinity chromatography.The expression and purification of the target protein were analyzed by SDS-PAGE and verified by western blot.The results showed that the recombinant purified MAP_2739 protein(rMAP_2739)with inclusion body expression was successfully obtained.The rabbit polyclonal antibodies against this protein were prepared,and the serum titer was 1∶51200 determined by ELISA.The MAP reference strain fraction was isolated by differential centrifugation,and the target bands appeared at 35ku in the cell wall,cell membrane and cell pulp,as verified by western blot using this polyclonal antibody as primary antibody,indicating that MAP_2739 is an extracellular protein.The enzymatic activity was determined by using p-nitrophenyl esters(p-NPs)as substrate,and the validation proved that MAP_2739 protein was lipase,which could hydrolyze long-chain fatty acids with the optimal enzymatic reaction pH value at 9.0,and rMAP_2739 protein still had lipase activity after pretreatment by pasteurization.The recombinant plasmid pET-22b-map_2739 gene was used as template,amplified by PCR using site-directed mutagenesis primers,verified by sequencing,transformed into E.coli expression strain,purified by western blot to verify protein expression,and analyzed by enzyme activity assay,respectively.The results showed that the purified proteins were successfully obtained for each point mutation,and S46 and D204 were identified as the enzyme active sites of MAP_2739.To sum up,this study demonstrated for the first time that MAP_2739 is a MAP extracellular GDSL lipase,which laid a foundation for further study on the pathogenesis of MAP.