基于杂合肽H5LL活性的生物信息学分析和重组乳酸菌表达载体的构建  

Bioinformatics analysis based on activity of heterotrimeric peptide H5LL and construction of recombinant Lactic acid bacteria expression vector

作　　者：赵潇颖 宿建胜 马嘉辉 王岳峰 王丹 孙丽媛 ZHAO Xiaoying;SU Jiansheng;MA Jiahui;WANG Yuefeng;WANG Dan;SUN Liyuan(Department of Clinical Biochemistry and Molecular Biology Testing,School of Medical Technology,Beihua University,Jilin 132013,China)

机构地区：[1]北华大学医学技术学院临床生化及分子生物学检验教研室,吉林吉林132013

出　　处：《吉林大学学报（医学版）》2023年第5期1366-1374,共9页Journal of Jilin University：Medicine Edition

基　　金：吉林省科技厅科技发展计划项目(2023020259NC,20230204085YY);北华大学研究生创新项目(北华研创合字[2021] 025)。

摘　　要：目的:以人源性抗菌肽(AMPs)富组蛋白5 (Hst5)和LL-37为亲本,设计新型杂合肽H5LL,并通过基因工程技术构建重组乳酸菌表达载体,实现AMPs的高效和安全表达。方法:采用生物信息学方法对H5LL进行理化参数、亲/疏水性、剪切位点、磷酸化位点、信号肽、跨膜区域、亚细胞定位和二级结构预测;将目的序列和空载质粒pMG36e经HindⅢ及KpnⅠ双酶切后,构建乳酸菌重组表达载体pMG36e-H5LL,克隆转化获得含目的基因的重组质粒。结果:H5LL成为AMPs的可能性较高,含36个氨基酸,相对分子质量为4 625 380,总电荷数为+10,具有亲水性;有3个磷酸化位点,无糖基化位点;属于膜内蛋白,无跨膜区域,无信号肽;亚细胞定位预测,线粒体靶向肽的可能性为0.333。二级结构中α-螺旋结构和β-转角占比分别52.78%及22.22%,三级结构中α-螺旋数量较多。含目的基因的重组质粒PCR电泳,于138 bp处有单一特异性条带;双酶切电泳,重组质粒在136和3 500 bp处有清晰条带。结论:设计并合成的新型杂合肽H5LL具有较高稳定性、抑菌活性和低毒性;成功构建重组乳酸菌表达载体pMG36e-H5LL。Objective:The human-derived antimicrobial peptide(AMPs)histatin 5(Hst5)and LL-37 were used as the parents to design a novel heterozygous peptide H5LL,and to construct a recombinant lactic acid bacteria expression vector by genetic engineering technology to achieve the efficient and safe expression of the AMPs.Methods:Bioinformatics method was used to predict the physicochemical parameters,hydrophilicity/hydrophobicity,shear sites,phosphorylation sites,signal peptides and transmembrane regions,subcellular localization,and secondary structure of H5LL;the target sequence and the empty plasmid pMG36e were doubly digested by HindⅢand KpnⅠ,then the lactic acid bacteria recombinant expression vector pMG36e-H5LL was constructed and cloned to obtain the recombinantplasmid containing the target genes. Results:H5LL had a high possibility of being an AMPs , contained36 amino acids, the relative molecular weight was 4 625 380,the total charge number was +10, which hadhydrophilicity;there were 3 phosphorylation sites and there was no glycosylation site;H5LL belonged tointramembrane protein, without transmembrane region and signal peptide;the subcellular localizationprediction results showed that the possibility of mitochondrial targeting peptide was 0. 333. The α -helixjunction and β-turned angle accounted for 52. 78% and 22. 22% in the secondary structure, and the numberof α-helix was the highest in the tertiary structure. The PCR electrophoresis results of recombinant plasmidcontained target gene showed a single specific band at 138 bp;the recombinant plasmid double digestionelectrophoresis results showed the clear bands at 136 and 3 500 bp. Conclusion:The novel heterogeneouspeptide H5LL is designed and synthesized with high stability, antibacterial activity and low toxicity;therecombinant lactic acid bacteria expression vector pMG36e-H5LL is successfully constructed.

关 键 词：富组蛋白5 抗菌肽 生物信息学 乳酸菌 表达载体 

分 类 号：Q939.9[生物学—微生物学]