牙龈卟啉单胞菌c-di-AMP代谢相关基因的克隆及表达纯化

目的 克隆牙龈卟啉单胞菌（Porphyromonas gingivalis）中负责c-di-AMP代谢的相关基因，并使其在大肠杆菌中正确表达及高效纯化。方法 通过聚合酶链反应（PCR）克隆牙龈卟啉单胞菌ATCC33277的pgn0523、pgn1187和pgn2003三个基因，经过酶切后将目的基因片段与大肠杆菌表达质粒pET28a连接，分别构建出重组表达质粒pETpgn0523 pETpgn0523、pET-pgn1187和pET-pgn2003。将重组表达质粒转化到大肠杆菌BL21（DE3）中，用异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达重组蛋白，通过十二烷基磺酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）分析和鉴定。用镍离子金属亲和层析法对重组蛋白进行分离纯化，BCA法检测目的蛋白的浓度。结果 目的基因pgn0523、pgn1187和pgn2003扩增产物条带与预期大小一致。重组表达质粒pET-pgn0523、pET-pgn1187和pET-pgn2003及其PCR产物琼脂糖凝胶电泳验证与预期大小相符，测序结果与GenBank中牙龈卟啉单胞菌ATCC33277国际标准菌株的基因序列100%一致。IPTG诱导后的菌体经SDS-PAGE检测得到3个大小分别为19.5×10³、39.9×10³、66.0×10³的蛋白质增强条带，与预期大小相符。镍离子金属亲和层析柱纯化得到的蛋白与预期目的蛋白分子量一致，BCA法测定目的蛋白的浓度分别为0.708、0.523和0.861 mg·mL^-1。结论 本研究成功克隆并表达纯化了牙龈卟啉单胞菌c-di-AMP代谢相关蛋白，得到了较高浓度和纯度的目的蛋白，为进一步探究牙龈卟啉单胞菌c-di-AMP的生理功能及体内代谢途径奠定了基础。

Cloning,expression, and purification of c-di-AMP metabolism-related genes from Porphyromonas gingivalis

Objective To clone, express, and purify cyclic diadenosine monophosphate (c-di-AMP) metabolism-related genes from Porphyromonas gingivalis (P. gingivalis) ATCC33277. Methods Polymerase chain reaction (PCR) from the genome of P. gingivalis ATCC33277 amplified the coding regions of pgn0523, pgn1187, and pgn2003 genes. The amplified DNA fragments were ligated with a prokaryotic expression vector pET28a to construct the recombinant expression plasmids pET-pgn0523, pET-pgn1187, and pET-pgn2003. These recombinant plasmids were transformed into Escherichia coli ( E. coli) BL21 (DE3) competent cells. The expression of recombinant proteins was induced by isopropyl-β-D-thiogalactoside and detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were purified using a Ni²⁺ matrix column, and their concentrations were determined by a BCA Protein Quantitative Kit. Results The c-di-AMP metabolism-related genes from P. gingivalis ATCC33277 were amplified successfully with the correct molecular size. The recombinant expression vectors were constructed by ligating enzyme-digested PCR products and pET28a vector, and verified by PCR and sequencing. After induction and purification, recombinant proteins were expressed successfully and obtained with the correct molecular size (19.5×10³, 39.9×10³, 66.0×10³). The final protein concentrations were 0.708, 0.523, and 0.861 mg·mL^-1 after dialysis. Conclusion The c-di-AMP metabolism-related genes from P. gingivalis ATCC33277 are cloned successfully, and their coding products are expressed correctly in E. coli. Highpurity proteins are finally obtained. The cloning and purification of these important proteins will help us to further investigate the physiological function and regulatory mecha-nism of c-di-AMP signaling system in P. gingivalis.