肺炎链球菌自溶素LytA小片段免疫活性的研究  

Immunological Activity of a Mini-Fragment of Pneumococcal Autolysin LytA

作　　者：邹琴[1] 杨光[1] 罗红[1] 宫晓红[2] 孙文平[1] 

机构地区：[1]大连医科大学临床免疫与微生物教研室,辽宁大连116044 [2]大连市中医医院,辽宁大连116013

出　　处：《微生物学杂志》2014年第3期52-55,共4页Journal of Microbiology

基　　金：辽宁省自然科学基金(201202053)

摘　　要：通过对肺炎链球菌（ATCC49619）自溶素lytA全序列基因的克隆和表达，获得部分小片段蛋白，初步研究其免疫活性。根据GenBank中肺炎链球菌M66菌株lytA基因序列（FN549899.1）设计合成特异性引物，采用PCR技术从肺炎链球菌（ATCC49619）基因组中扩增lytA全序列片段，构建克隆载体PGM T/lytA, 测序后与M66菌株比对显示，肺炎链球菌ATCC49619株碱基序列内存在新酶切位点BamHⅠ。以BamHⅠ、HindⅢ双酶切后，目的基因出现2个小片度，以约500 bp小片段构建重组表达载体pET32a（＋）/lytA′，经IPTG诱导后等电点洗脱法获取纯化的目的蛋白LytA′。将LytA′免疫小鼠，测定抗体效价，同时进行Western blot鉴定。测序显示肺炎链球菌ATCC49619菌株与M66菌株的lytA全基因序列同源性为98%，具有高度保守性，碱基不同主要位于下游部分，在444～450 bp之间新出现BamHⅠ酶切位点。构建的小片段重组表达质粒pET32a（＋）/lytA′在BL21（DE3）中高效表达，获得纯化蛋白LytA′，免疫BALB/c小鼠产生的抗体效价为1：32，经Western blot证实该蛋白具有较好的抗体结合活性，为进一步应用于疫苗及药物等相关研究奠定基础。A part fragment of protein obtained from cloning and expressing the whole sequence of autolysin LytA of Streptococcus pneumonia ATCC49619 was studied initially for its immune activity. A specific primer were designed according to LytA gene sequence （FN549899.1） of S. pneumonia （S. pn） M66 strain in Genbank, and the lytA gene was amplified by PCR from S. pn strain （ATCC49619）. The recombinant vector PGM T/LytA was constructed. After sequencing it was displayed and compared with M66 strain, in LytA of ATCC49619 strain there was a new restriction site BamHI. After digested with double enzymes of BamHI and HindIII, the goal gene emerged two mini fragments, and constructed and expressed at about 500 bp mini fragment of recombinant vector pET32a（＋）/LytA′. And transformed into BL21（DE3） to expressed protein LytA′ after induced by IPTG and isoelectric elution to obtain purified the goal protein LytA′. The antibody valance of serum from BALB/c mice immunized with LytA′protein was detected by double diffusion test. Reactivity of the protein LytA′ with the accordingly antibody was studied by Western blot. The homology rate of LytA gene sequence between S. pn ATCC49619 strain and S. pn M66 strain was 98%, with high degree of conservativeness, the base difference mainly located on downstream, and a new BamHI restriction site emerged on 444~450 bp. The recombinant protein LytA′ was expressed and purified successfully with a relative molecular weight of 20 ku. The valance of antibody from mice immunized with protein LytA′ was 1：32. Western blot test showed that the protein had a fine antibody combining activity, and it has laid a foundation for further application in vaccine and medicine and other related studies.

关 键 词：肺炎链球菌 自溶素 酶切 免疫活性 

分 类 号：Q939.91[生物学—微生物学]