吉林大学学报(医学版) ›› 2023, Vol. 49 ›› Issue (5): 1366-1374.doi: 10.13481/j.1671-587X.20230534

• 方法学 • 上一篇    

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

赵潇颖,宿建胜,马嘉辉,王岳峰,王丹,孙丽媛()   

  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
  • 收稿日期:2022-12-03 出版日期:2023-09-28 发布日期:2023-10-26
  • 通讯作者: 孙丽媛 E-mail:jlsunliyuan@163.com
  • 作者简介:赵潇颖(1993-),女,吉林省吉林市人,在读硕士研究生,主要从事病原微生物和抗菌肽方面的研究。
  • 基金资助:
    吉林省科技厅科技发展计划项目(2023020259NC);北华大学研究生创新项目(北华研创合字〔2021〕025)

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

Xiaoying ZHAO,Jiansheng SU,Jiahui MA,Yuefeng WANG,Dan WANG,Liyuan SUN()   

  1. Department of Clinical Biochemistry and Molecular Biology Testing,School of Medical Technology,Beihua University,Jilin 132013,China
  • Received:2022-12-03 Online:2023-09-28 Published:2023-10-26
  • Contact: Liyuan SUN E-mail:jlsunliyuan@163.com

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。

关键词: 富组蛋白5, 抗菌肽, 生物信息学, 乳酸菌, 表达载体

Abstract:

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 HindⅢ and KpnⅠ, then the lactic acid bacteria recombinant expression vector pMG36e-H5LL was constructed and cloned to obtain the recombinant plasmid containing the target genes. Results H5LL had a high possibility of being an AMPs, contained 36 amino acids, the relative molecular weight was 4 625 380,the total charge number was +10, which had hydrophilicity; there were 3 phosphorylation sites and there was no glycosylation site; H5LL belonged to intramembrane protein, without transmembrane region and signal peptide;the subcellular localization prediction results showed that the possibility of mitochondrial targeting peptide was 0.333. The α-helix junction and β-turned angle accounted for 52.78% and 22.22% in the secondary structure, and the number of α-helix was the highest in the tertiary structure. The PCR electrophoresis results of recombinant plasmid contained target gene showed a single specific band at 138 bp; the recombinant plasmid double digestion electrophoresis results showed the clear bands at 136 and 3 500 bp. Conclusion The novel heterogeneous peptide H5LL is designed and synthesized with high stability, antibacterial activity and low toxicity;the recombinant lactic acid bacteria expression vector pMG36e-H5LL is successfully constructed.

Key words: Histatin5, Antimicrobialpeptides, Bioinformatics analysis, Lactic acid bacteria, Expression vector

中图分类号: 

  • Q939.9