遗传 ›› 2019, Vol. 41 ›› Issue (8): 736-745.doi: 10.16288/j.yczz.19-087

• 研究报告 • 上一篇    下一篇

微生物源果胶酶在猪PK15细胞中异源表达及其酶学性质分析

莫健新1,王豪强2,黄广燕2,蔡更元1,2,吴珍芳1,2,张献伟1,2()   

  1. 1. 温氏食品集团股份有限公司,新兴 527400
    2. 华南农业大学国家生猪种业工程技术研究中心,广州 510642
  • 收稿日期:2019-03-27 修回日期:2019-07-18 出版日期:2019-08-20 发布日期:2019-08-01
  • 通讯作者: 张献伟 E-mail:zxianw@163.com
  • 作者简介:莫健新,硕士研究生,专业方向：动物遗传育种与繁殖。E-mail:2696064146@qq.com
  • 基金资助:
    国家转基因重大专项(2016ZX08006002);广东省科技厅平台条件建设项目资助(2013B070702003)

Heterogenous expression and enzymatic property analysis of microbial-derived pectinases in pig PK15 cells

Jianxin Mo1,Haoqiang Wang2,Guangyan Huang2,Gengyuan Cai1,2,Zhenfang Wu1,2,Xianwei Zhang1,2()   

  1. 1. Wens Foodstuff Group Co. LTD, Xinxing 527400, China
    2. National Engineering Research Center for Swine Breeding Industry, South China Agricultural University, Guangzhou 510642, China
  • Received:2019-03-27 Revised:2019-07-18 Online:2019-08-20 Published:2019-08-01
  • Contact: Zhang Xianwei E-mail:zxianw@163.com
  • Supported by:
    Supported by the National Transgenic Major Projects(2016ZX08006002);Guangdong Provincial Science and Technology Department Platform Condition Construction Project(2013B070702003)

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摘要：

果胶是植物细胞壁组分之一,是畜禽饲料中主要的抗营养因子,影响畜禽对日粮中能量和氮的利用效率。果胶酶在自然界中广泛存在于细菌、酵母和丝状真菌等微生物中,对解除果胶的抗营养作用、提高饲料利用率具有良好的效果。为了探索在猪细胞中表达微生物源果胶酶基因的可行性,本研究通过脂质体转染法将微生物源果胶酶基因pg5a、pgI、pga3A和pgaA导入猪PK15细胞中进行异源表达,利用3,5-二硝基水杨酸(3,5-dinitrosalicylic acid, DNS)法测定这4种基因编码果胶酶的酶活力。结果显示,4种果胶酶基因均能在猪PK15细胞中转录出mRNA,但只有pg5a和pgI适应猪细胞表达系统。其中,果胶酶PG5A的最高酶活为0.95 U/mL,最适作用pH为pH4.0,在pH4.6~6.0范围内维持46%以上的酶活;PGI在pH5.0处获得最高酶活,为0.30 U/mL,在pH4.0~6.0范围内维持35%以上的酶活。消化蛋白酶耐受实验结果显示,PG5A和PGI对胃蛋白酶和胰蛋白酶均有较强的耐受能力,用1 mg/mL的猪胃蛋白酶处理2 h后,PG5A和PGI的剩余酶活分别为76%和71%;用1 mg/mL的猪胰蛋白酶处理2 h后,PG5A和PGI的剩余酶活分别为44%和93%。综上所述,果胶酶基因pg5a和pgI能在猪细胞中正常表达,其编码的果胶酶对猪消化道pH条件和消化蛋白酶具有较强的耐受能力,可作为制备转果胶酶基因猪的候选基因。

关键词: 果胶酶, 果胶, 抗营养因子, 转基因, 猪

Abstract:

As one of plant cell wall components, pectin is the main anti-nutritional factor in livestock and poultry feeds and has an adverse effect on utilization efficiency of feed energy and nitrogen. Pectinases, which are widely found in microorganisms such as bacteria, yeast and filamentous fungi in nature,can improve feed efficiency by relieving the anti-nutritional effect of pectin through promoting the hydrolysis reaction of feed pectin. To explore the feasibility of expressing microbial-derived pectinase genes in pig cells, we introduced microbial-derived pectinase genes pg5a, pgI, pga3A, and pgaA into porcine PK 15 cells by lipofection for heterogenous expression. Enzymatic activities of the pectinases encoded by these genes were analyzed using the 3,5 dinitrosalicylic acid (DNS) method. Results showed that all four pectinase genes were able to be transcribed into mRNAs in porcine PK 15 cells, but only pg5a and pgI were adapted to the porcine cell expression system. Among them, the maximum activity of pectinase PG5A was 0.95 U/mL, the optimum pH was pH 4.0, and the enzymatic activity was maintained above 46% in the range of pH 4.6 to 6.0. Pectinase PGI obtained the highest enzymatic activity at pH 5.0, which was 0.30 U/mL, and maintained more than 35% of the activity in the range of pH 4.0 to 6.0. The results of digestive protease tolerance test showed that PG5A and PGI were highly resistant to pepsin and trypsin. After treatment with 1 mg/mL pig pepsin for two hours, the residual enzymatic activities of PG5A and PGI were 76% and 71%, respectively. And after two hours treatment with 1 mg/mL of pig trypsin, the remaining enzymatic activities of PG5A and PGI were 44% and 93%, respectively. In summary, pectinase PG5A and PGI can be effectively expressed in pig cells, and have strong tolerance to pig intestinal pH environment and digestive proteases. Therefore, both pg5a and pgI can be used as candidate genes for production of transgenic pigs.

Key words: pectinases, pectin, anti-nutritional factors, transgene, pigs