重组鱼精蛋白的制备及抑菌活性分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-0076

摘要/Abstract

摘要：

旨在利用原核表达系统实现重组鱼精蛋白的表达,并检测其生物学活性。利用基因工程手段将CYGB中甲硫氨酸位点ATG（除起始密码子）全部突变为ATC获得CYGBm。采用CYGBm与鱼精蛋白融合方法表达重组鱼精蛋白,CNBr裂解CYGBm与鱼精蛋白中的甲硫氨酸位点,并得以纯化。采用试管培养法分析重组鱼精蛋白抑菌活性,鲜虾检测其应用效果。CYGBm与鱼精蛋白融合可以在原核表达系统中大量以包涵体形式表达,经分离与CNBr裂解制备了纯化的重组鱼精蛋白,经验证重组鱼精蛋白具有很好的抑菌活性。采用基因工程方法可以实现具有抑菌活性重组鱼精蛋白的制备,为工业化生产重组鱼精蛋白提供理论基础。

关键词: 鱼精蛋白, 突变, 融合, 抑菌

Abstract:

This work aims to express recombinant protamine by prokaryotic expression system and to detect its biological activity. First,genetic engineering was used to obtain CYGBm by mutating all the methionine ATG sites（except the start codon）into ATC. Then,CYGBm and protamine fusion method was applied to express the recombinant protamine. CNBr was employed to cleave the methionine sites in the CYGBm and protamine and thus it was purified. Finally,tube culture method was to analyze the antimicrobial activity of the recombinant protamine and its application effect was detected by fresh shrimp. As results,the fusion of the CYGBm and the protamine could be expressed in a large number of inclusion bodies in the prokaryotic expression system. The purified recombinant protamine was prepared by separation and CNBr lysis. It was verified that the recombinant protamine demonstrated excellent antibacterial activity. In conclusion,the preparation of recombinant protamine with antibacterial activity can be achieved by genetic engineering method,which provides a theoretical basis for industrial production of recombinant protamine.

Key words: protamine, mutation, fusion, bacteriostatic

李招发, 邱丹丹. 重组鱼精蛋白的制备及抑菌活性分析[J]. 生物技术通报, 2021, 37(10): 162-168.

LI Zhao-fa, QIU Dan-dan. Preparation of Recombinant Protamine and Analysis of Its Antibacterial Activity[J]. Biotechnology Bulletin, 2021, 37(10): 162-168.

图/表 7

表1 本研究涉及的寡核苷酸序列

Table 1 Oligonucleotide sequences involved in this study

Oligonucleotide name	Sequence（5'-3'）
SEQ ID NO.1	AACATATGGAGAAAGTGCCAGGCGAG
SEQ ID NO.2	GGCCCAGATAGCCTGCACCGCCTTCC
SEQ ID NO.3	CGGTGCAGGCTATCTGGGCCCGGCTC
SEQ ID NO.4	CTCCAGGGGATCTTCGATGTGCTTGAACTGGC
SEQ ID NO.5	CACATCGAAGATCCCCTGGAGATCGAGCGGAGC
SEQ ID NO.6	GAGGGCCCCGATGACTCGGCAGGCGTGC
SEQ ID NO.7	GCCGAGTCATCGGGGCCCTCAACAC
SEQ ID NO.8	CAGGATCCCGGCCCCGAAGAGGGCAGTG

图1 重叠延伸PCR定点突变CYGB流程图

Fig. 1 Flow chart of fixed point mutations CYGB by overlapping extended PCR

图2 CYGB和CYGBm全菌SDS-PAGE

Fig. 2 Whole strains SDS-PAGE of CYGB and CYGBm M:Marker. 1:CYGB. 2:CYGBm

图3 重组质粒pET22b-CYGBm-Pro PCR法和双酶切法核酸电泳图 M1: 15 000 bp标准分子量DNA; 1: BL21菌落PCR; 2: BL21-pET22b-CYGBm-Pro菌落PCR; 3: pET22b-CYGBm-Pro质粒Nde I/EcoR I双酶切; M2: 2 000 bp标准分子量DNA

Fig. 3 Nucleic acid electrophoresis of recombinant plasmid pET22 B-CYGBm-Pro by PCR and double enzyme digestions M1:15 000 bp marker DNA. 1:PCR of BL21 bacterial colony. 2:PCR of BL21-pET22b-CYGBm-Pro bacterial colony. 3:Plasmid of pET22b-CYGBm-Pro by double enzyme digestions of Nde I/Eco I. M2:2 000 bp marker DNA

图4 CYGBm-Pro表达以及纯化的CYGBm-Pro和rPro的SDS-PAGE图 M1 :标准分子量蛋白; 1 :末诱导全菌 ;2 :乳糖诱导全菌; 3:乳糖诱导全菌裂解液上清 ;4 :乳糖诱导全菌裂解液沉淀;5:纯化的细胞珠蛋白融合的鱼精蛋白;6:纯化的重组鱼精蛋白

Fig. 4 SDS-PAGE of CYGBm-Pro expression and purified CYGBm-Pro and rPro M1:marker. 1:Uninduced whole bacterium. 2:Induced whole bacterium. 3:Supernatant of induced whole bacterium. 4:Sediment of induced whole bacterium. 5:Purified CYGB-Pro. 6:Purified rPro

图5 不同浓度rPro对不同细菌生长抑制率曲线图

Fig. 5 Curves of growth inhibition rates of different bacteria with different concentrations of rPro

图6 rPro处理虾储存期间pH值（A）和TVB-N值（B）变化曲线图

Fig. 6 Curves of pH values（A）and TVB-N values（B）of shrimp treated by rPro during storage

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