金黄色葡萄球菌表面蛋白A对奶牛乳腺上皮细胞的黏附作用

本试验拟探究金黄色葡萄球菌（金葡菌）表面蛋白A（Staphylococcus aureus surface protein A，SasA）编码基因在奶牛乳源金葡菌中是否具有普遍性及同源性，并结合蛋白结构组成研究SasA对奶牛乳腺上皮细胞的黏附作用。试验前期从中国北方5个荷斯坦牛场无菌条件下分离纯化了73株奶牛乳源金黄色葡萄球菌菌株，PCR扩增鉴定SasA基因并进行序列保守性分析。利用原核表达系统表达SasA蛋白的丝氨酸富集片段1（serine-rich repeat region 1， SRR1）及非重复区域（non-repeat region， NRR）并进行蛋白纯化。利用流式细胞仪检测NRR，牛血清白蛋白（bovine serum albumin， BSA）及SRR1对奶牛乳腺上皮细胞（Mac-T）黏附性差异。为进一步在NRR中定位发挥主要黏附作用的片段，试验采用了长度不同的NRR片段与完整NRR片段做竞争性黏附处理。PCR扩增产物鉴定及序列同源性分析结果显示，86.3%的牛源金葡菌菌株含SasA基因且序列一致性超过95%。相较于SRR1和BSA，NRR对细胞具有更强的黏附性。黏附抑制试验结果表明，NRR1-2片段（230—540 aa）对NRR抑制作用最明显。综上表明，SasA基因在奶牛乳源金葡菌中具有普遍性，该基因序列具有高度的保守性。在SasA蛋白中，对奶牛乳腺上皮细胞起主要黏附作用的为NRR1-2片段，大致定位在其结构域的230—540位氨基酸。本研究结果表明，与该区域结合的受体中可能存在SasA作为黏附素与奶牛乳腺上皮细胞互作的位点。

The Adhesion Effect of Staphylococcus aureus Surface Protein A on Dairy Cattle Mammary Epithelial Cells

This study was conducted to explore the universality and homology of the sequence encoding Staphylococcus aureus surface protein A (SasA) in S. aureus strains existing in raw milk. Combined with the investigation into the structure and composition, we can elucidate its role in adhesion to dairy cattle mammary epithelial cells. Seventy-three S. aureus strains were isolated and purified from five dairy cattle farms in the north of China using aseptic techniques. After DNA extraction, we used PCR to amplify and identify SasA gene, then compared the sequence with reference sequences to analyze its conservation. SRR1(serine-rich repeat region 1) and NRR(non-repeat region)were reconstructed using prokaryotic expression system and purified. The differences in adhesion effect of NRR, BSA and SRR1 on dairy cattle mammary epithelial cells (Mac-T) were detected by flow cytometer. Compared with BSA and SRR1, NRR exhibits greater adhesion to cells. To find out the main adhesion domain in NRR, different lengths of NRR fragments were used to inhibit the intact NRR fragment adhesion. PCR combined with the homologous analysis of sequences showed 86.3% of S. aureus carried SasA gene and the gene similarity was over 95% with the adhesion effect of NRR1-2 (230-540 aa) being most obvious. The above results have indicated that SasA gene is ubiquitous and highly conserved in bovine S. aureus. NRR1-2 module plays a major role in adhesion to dairy cattle mammary epithelial cells and is roughly located at the 230-540th residues of the protein domain. It suggests that specific cell receptors, which can bind to this module, might be sites where SasA interacts with the host cells as a kind of adhesion.