中文摘要：

目的：通过基因工程方法获得重组的杀白细胞毒素，为深入研究其致病机制打下基础。方法：利用PCR方法从产杀白细胞毒素金葡菌临床分离株的基因组DNA中克隆PVL—S和PVL—F基因序列，分别将其克隆到载体pET22b中，构建重组pET22b—LuKS和pET22b—LuKF原核表达载体，转化感受态大肠杆菌BL21（DE3）pLysS，异丙基硫代β—D半乳糖苷（sopropyl—beta—D—thiogalactopyranoside，IPTG）诱导表达，将其可溶性表达经镍离子螯合亲和层析纯化后，通过对外周血粒细胞和多形核白细胞的刺激反应鉴定其活性。结果：以PCR方法获得PVL—S和PVL—F基因片段，与克隆载体连接后测序与文献报道的基本一致，成功构建了pET22b—LuKS和pET22b—LuKF原核表达质粒，并高效诱导表达出相对分子质量约34kD的S蛋白及相对分子质量约35kD的F蛋白。重组蛋白（rPVS，rPVF）在37℃经IPTG诱导时均以包涵体形式存在，而30℃时部分出现可溶性表达，部分为包涵体。将上清可溶性重组蛋白作用于外周血粒细胞，可导致粒细胞出现凋亡，部分出现坏死。结论：成功构建了表达载体pET22b—LuKS和pET22b—LuKF，对其编码的S片段和F片段进行了原核表达和鉴定，高效表达了杀白细胞毒素的S和F两类蛋白，为进一步对其致病机制和免疫原性的研究奠定基础。

英文摘要：

AIM： Panton - Valentine leukocidin （PVL） is a pore - forming toxin secreted by Staphylococcus aureus epidemiologically associated with the often - lethal necrotizing pneumonia. Until now, the mechanisms of pathogenesis of PVL leading to the fatal pulmonia remains undefined and also acquired plenty of the toxins is difficult. In the present study, we obtain recombinant staphylococcal F and S components of the Panton - Valentine leukocidin by gene engineering and evaluate its biological activity in vitro, which provides an experimental basis for the further studies of its biological function and its toxicity in pneumonia. METHODS ： The full - length of F and S components of PVL gene amplified from the strain of Staphylococcus aureus DNA by high - fidelity PCR was cloned into prokaryotic expression vector pET22b（ ＋ ）, and the vector was transformed into BI21 （DE3）plysS to construct a prokaryotic expression system. The integrity of the opening -reading frame of each construct was verified by DNA sequencing. The recombinant PVL （rPVL） was induced by1. 0 mmol/L IPTG. The expressed products were identified by SDS - PAGE and the fusion proteins （6His - LukS - PV and 6His - LukF - PV） were purified from lysates of transfected E. coli cells by affinity chromatography on nitrilotriacetic acid columns. The cytolytic activity was tested by incubation of rPVL with human polymorphonuclear neutrophils （PMNs） in vitro. RESULTS： The nucleotide sequence of the cloned PVL gene was the same as that of reported in GenBank. E. coli BI221 （DE3）plysS containing recombinant vectos grow at 37℃ causes some proteins to accumulate as inclusion bodies, while incubation at 30℃ led to a significant amount of soluble active proteins which accounted for about 31.7% of the total bacterial protein. The relative molecular weight showed on SDS - PAGE profile was consistent with the expected value which the LukS - PV protein was about 34 kD, and the LukF - PV protein was about 35 kD. The purified rPVL was obtaine