融合抗菌肽基因在重组毕赤酵母的表达及体外活性研究 <sup>*</sup>

doi:10.13523/j.cb.20180602

中国生物工程杂志

2018, Vol. 38

Issue (6): 9-16 DOI: 10.13523/j.cb.20180602

研究报告

融合抗菌肽基因在重组毕赤酵母的表达及体外活性研究 ^*

唐健雪,肖永乐,彭俊杰,赵世纪,万小平,高荣(

)

四川大学生命科学学院生物资源与生态环境教育部重点实验室四川省动物疫病预防与食品安全重点实验室成都 610065

Expression of Fusion Antibacterial Peptide in Recombinant Pichia pastoris and Its Bioactivity In Vitro

Jian-xue TANG,Yong-le XIAO,Jun-jie PENG,Shi-ji ZHAO,Xiao-ping WAN,Rong GAO(

)

Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610065, China

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摘要：

目的:在毕赤酵母SMD1168中表达融合抗菌肽,并检测其体外抑菌活性。方法:本实验从实验室先前构建的重组质粒pVAX1-RHKJT中克隆出已构建好的融合抗菌肽RHKJT基因片段,将RHKJT基因片段插入至pGAPZaA真核表达质粒中,通过PCR和测序验证,构建pGAPZα-RHKJT重组真核表达质粒,将线性化的pGAPZα-RHKJT电转化至毕赤酵母SMD1168中获得重组酵母SMDpG-RHKJT,并通过PCR和RT-PCR验证,对重组毕赤酵母SMDpG-RHKJT进行发酵,并收集发酵上清液进行体外生物活性测定。结果: 成功获得重组酵母SMDpG-RHKJT菌株,重组酵母发酵上清液对大肠杆菌标准菌、大肠杆菌耐药菌、沙门氏菌标准菌、金黄色葡萄球菌标准菌、金黄色葡萄球菌耐药菌、肺炎链球菌标准菌均具有显著的抑菌活性。结论:重组酵母表达的融合抗菌肽具有较广的抗菌谱和较高的抑菌活性,具有良好的潜在应用前景。

关键词： 融合抗菌肽; 基因表达; 毕赤酵母; 抑菌生物活性

Abstract:

Objective:To express fusion antimicrobial peptide in Pichia pastoris SMD1168 and determine its in vitro bioactivity.Methods:The constructed fusion anti-peptide RHKJT gene fragment was cloned from the recombinant pVAX1-RHKJT vector previously constructed in laboratory. The RHKJT gene fragment was inserted into plasmid pGAPZaA, then verified by PCR and sequenced to construct recombinant pGAPZα-RHKJT vector. The linearized pGAPZα-RHKJT was electroporated into Pichia pastoris SMD1168 to obtain the recombinant yeast SMDpG-RHKJT. The recombinant Pichia pastoris SMDpG-RHKJT was fermented and confirmed by PCR and RT-PCR. Fermentation supernatants were collected for in vitro bioactivity assay.Results:The recombinant yeast SMDpG-RHKJT was successfully obtained, and the recombinant yeast’s fermentation supernatant had significant inhibition effects on Escherichia coli, Salmonella, Staphylococcus aureus and Streptococcus pneumonia.Conclusion:The recombinant antimicrobial peptide expressed by recombinant yeast has marked antibacterial activity and would facilitate the development of novel antibacterial additive feed later.

Key words: Fusion antibacterial peptide Gene expression Recombinant Pichia pastoris bacteriostasis Antibacterial bioactivity

收稿日期: 2018-01-02 出版日期: 2018-07-06

ZTFLH:

Q786

基金资助: * 四川省国际科技合作(2017HH0023);成都市科技攻关(2015NY-02-0028-NC);四川省科技(2016RZ0034);科技部国际合作资助项目(2011DFA10101103)

通讯作者: 高荣 E-mail: gaorong96@163.com

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引用本文:

唐健雪,肖永乐,彭俊杰,赵世纪,万小平,高荣. 融合抗菌肽基因在重组毕赤酵母的表达及体外活性研究 ^*[J]. 中国生物工程杂志, 2018, 38(6): 9-16.

Jian-xue TANG,Yong-le XIAO,Jun-jie PENG,Shi-ji ZHAO,Xiao-ping WAN,Rong GAO. Expression of Fusion Antibacterial Peptide in Recombinant Pichia pastoris and Its Bioactivity In Vitro. China Biotechnology, 2018, 38(6): 9-16.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180602 或 https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I6/9

表1 引物序列

表2 六种细菌菌株的抗生素使用浓度梯度

图1 PCR扩增得到的融合抗菌肽片段的电泳图(1%琼脂糖凝胶)

图2 重组质粒pGAPZαA- RHKJT PCR 扩增产物电泳图 (1%琼脂糖凝胶)

图3 重组酵母菌的PCR鉴定(1%琼脂糖凝胶)

图4 RT-PCR电泳检测重组酵母转录情况(1%琼脂糖凝胶)

图5 大肠杆菌标准菌抑菌实验结果

图6 大肠杆菌耐药菌抑菌实验结果

图7 肺炎链球菌标准菌抑菌实验结果

图8 金黄色葡萄球菌标准菌抑菌实验结果

图9 金黄色葡萄球菌耐药菌抑菌实验结果

图10 沙门氏菌标准菌抑菌实验结果

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