绵羊抗菌肽GNLY基因多态性分析及合成多肽的生物学功能研究

摘要

试验旨在分析绵羊抗菌肽颗粒溶素(granulysin,GNLY)基因多态性,检测合成多肽的抑菌活性和抑制肿瘤细胞活性,明确绵羊抗菌肽GNLY的生物学功能.通过对 GNLY基因 RT-PCR产物进行测序,分析 GNLY基因多态性,推导其氨基酸序列信息合成功能区多肽,利用径向扩散试验和M IC试验检测合成多肽对大肠杆菌、沙门氏菌、葡萄球菌、金黄色葡萄球菌的抑制作用;利用M T T试验观察合成多肽对人食管癌细胞(EC109)、人肾癌细胞(X786-0)的抑制作用.结果发现,M IC试验中浓度>250 μg/mL的G16对大肠杆菌、葡萄球菌均有抑制作用;浓度≥7.82 μg/mL的GS16对大肠杆菌抑制作用明显,对沙门氏菌、葡萄球菌和金黄色葡萄球菌无明显抑制作用;62.5 μg/mL GC16对大肠杆菌、沙门氏菌和金黄色葡萄球菌均有抑制作用,其中对大肠杆菌和金黄色葡萄球菌抑制作用较为明显;G22对4种细菌均无明显的抑制作用.M T T试验结果表明,合成多肽G16、GS16对癌细胞无抑制作用,G22和GC16对EC109、X786-0具有显著抑制作用,其中1 mg/mL G22和1 mg/mL GC16对EC109生长抑制率分别为88.21% 和57.21%,对X786-0生长抑制率分别为96.37% 和81.87%.研究显示,合成GNLY多肽对细菌和肿瘤细胞具有一定的抑制活性,本研究结果为绵羊抗菌肽作为候选抗菌药物的开发奠定了基础.%This study was aimed to analyze the polymorphism of granulysin(GNLY)gene of sheep antimicrobial peptide,detect the antibacterial activity and inhibition tumor cell activity of synthetic peptides,and clarify the biological function of sheep antimicrobial peptide GNLY.In this study,the polymorphism of GNLY gene was analyzed by sequencing RT-PCR products of GNLY gene,and its deduced amino acid fragments were synthesized,the antibacterial effect of synthetic peptides on Escherichia coli,Salmonella,Staphylococcus and Staphylococcus aureus were deter-mined using radial diffusion test and MIC test,and its inhibition growth to human esophageal cancer cells(EC109)and human renal carcinoma cells(X786-0)were detected using MTT test.The results showed that the concentration of G16> 250 μg/mL in the MIC test inhibited the growth of Escherichia coli and Staphylococcus aureus;The concentration of GS16≥7.82 μg/mL obviously inhibited the grow th of Escherichia coli,there was no inhibitory effect on Salmonella, Staphylococcus and Staphylococcus aureus.62.5 μg/mL GC16 had inhibitory effect on all four strains(except for Staphylococcus)and obviously inhibited the growth of Escherichia coli and Staphylococcus aureus.G22 had no inhibitory effect on all four strains.G22 and GC16 obviously inhibited the grow th of EC109 and X786-0,the grow th inhibition rates against EC109 of 1 mg/mL G22 and 1 mg/mL GC16 were 88.21% and 57.21%,and its against X786-0 were 96.37% and 81.87%,respectively.G16 and GS16 had no inhibitory activity to tumor cells.This study indicated that the synthetic peptides of GNLY had inhibitory activity on bacteria and tumor cells,also laid the foundation for the development of sheep antibacterial peptides as candidate antibacterial medicine.