重组猪表皮生长因子在酿酒酵母中的克隆表达及其生物学活性鉴定

doi:10.11843/j.issn.0366-6964.2014.12.009

畜牧兽医学报 ›› 2014, Vol. 45 ›› Issue (12): 1971-1980.doi: 10.11843/j.issn.0366-6964.2014.12.009

重组猪表皮生长因子在酿酒酵母中的克隆表达及其生物学活性鉴定

王蜀金^1,3，周琳²，陈惠娜^1,3，张正帆¹，黄艳玲¹，郭春华^1*，王永^1,3

(1.西南民族大学生命科学与技术学院，成都 610041； 2.深圳比利美英伟营养饲料有限公司，深圳 518103；3.动物遗传育种学国家民委-教育部重点实验室，成都 610041）

收稿日期:2014-04-11 出版日期:2014-12-23 发布日期:2014-12-23
通讯作者: 郭春华，博士，教授，主要从事分子营养研究，E-mail:531893818@qq.com
作者简介:王蜀金（1988-），男，重庆人，硕士生，主要从事分子营养研究，E-mail：137638758@qq.com
基金资助:
国家科技支撑计划（2012BAD13B06）；西南民族大学研究生创新型科研项目（CX2013SZ44）

Cloning and Expression of Recombinant Pig Epidermal Growth Factor in Saccharomyces cerevisiae and Its Biological Activity

WANG Shu-jin^1,3，ZHOU Lin²，CHEN Hui-na^1,3，ZHANG Zheng-fan¹，HUANG Yan-ling¹，GUO Chun-hua^1*,WANG Yong^1,3

(1.College of Life Science and Technology，Southwest University for Nationalities，Chengdu 610041，China；2.Shenzhen Premix Nutrition CO.，LTD，Shenzhen 518103，China；3.Key Laboratory of Animal Genetics and Breeding，State Ethnic Affairs Commission and Ministry of Education，Chengdu 610041，China)

Received:2014-04-11 Online:2014-12-23 Published:2014-12-23

摘要/Abstract

摘要：

猪表皮生长因子（Porcine epidermal growth factor，pEGF）能够刺激小肠DNA合成及细胞增殖，促进肠道发育，从而提高断奶仔猪生产性能及免疫功能等。由此，研发分泌表达猪表皮生长因子的益生菌，用于畜牧生产及临床医学具有重要意义。克隆内江猪乳腺组织EGF及酿酒酵母中分泌信号肽Mfα，在综合考虑酿酒酵母密码子使用频率和表达效率的基础上，人工合成优化后的内江猪EGF基因（Synthetic porcine epidermal growth factor，spEGF），构建pYES2-Mfα-spEGF表达载体并在酿酒酵母中成功表达。该重组酿酒酵母INVSc1( pYES2-Mfα-spEGF) 表达蛋白spEGF进行Tricine-SDS-PAGE电泳、蛋白质免疫印迹、体外生物学活性检测及断奶SD大鼠体内生物学活性检测。根据RT-PCR和测序结果显示，成功地构建了pYES2-Mfα-spEGF表达载体并在酿酒酵母中实现表达；在Tricine-SDS-PAGE电泳及蛋白质免疫印迹中可以分别观察到目的条带及印迹条带；体外生物学活性检测结果显示，INVSc1( pYES2-Mfα-spEGF) 表达蛋白spEGF对肠上皮细胞具有明显增殖作用（P<0.05）；断奶SD大鼠体内生物学活性检测结果表明，重组酿酒酵母组INVSc1( pYES2-Mfα-spEGF) 能促进小肠发育（如绒毛高度、隐窝深度、小肠黏膜总蛋白、总DNA及RNA含量等）（P<0.05），显著提高其生产性能（如体重、日增重、采食量及饲料转化率）及免疫功能（如IgA、IgG及IgM）等（P<0.05）。本研究成功构建的INVSc1( pYES2-Mfα-spEGF) 菌液上清中目的蛋白的表达量约为30 mg•L^-1，且具有高生物学活性，可以直接用于畜牧生产及临床医学。

Abstract:

Porcine epidermal growth factor (EGF) can induce effects on DNA synthesis and stimulation of cellular proliferation in intestine to improve the performance，gut development and immune function of early weaned-piglets.Herein，generation of Neijiang pig EGF-expressing Saccharomyces cerevisiae (S.cerevisiae) was beneficial to livestock production and clinical research；pYES2-Mfα-spEGF were originated from synthetic optimized EGF from breast tissue of Neijaing pig (pEGF) and signal peptide (Mfα) fragments of S.cerevisiae were cloned into pYES2/CT.Recombinant S.cerevisiae INVSc1(pYES2-Mfα-spEGF) expressing EGF protein was generated by transforming the plasmid pYES2-Mfα-spEGF into S.cerevisiae INVSc1.The target EGF protein was determined by Tricine-SDS-PAGE electrophoresis and Western blot，meanwhile，its biological activity was also determined in vivo and in vitro.The spEGF-expressing recombinant S.cerevisiae INVSc1(pYES2-Mfα-spEGF) was constructed successfully and expressed in S.cerevisiae.The target protein EGF was identified by Tricine-SDS-PAGE electrophoresis and Western blot.The results of biological activity detection showed that spEGF-expressing protein could stimulate enterocyte proliferation (P<0.05)，improve the growth performance (e.g.，body weight，average daily gain，feed intake，and feed conversion rate) (P<0.05)，gut development (e.g.，villus height，crypt depth，and contents of total protein，DNA，and RNA) (P<0.05)，and immune function (e.g.，IgA，IgG，and IgM) of early-weaned SD rat (P<0.05).The concentration of target protein from the supernatant fluid of INVSc1(pYES2-Mfα-spEGF) is about 30 mg•L^-1，and INVSc1(pYES2-Mfα-spEGF) have some prominent features encompassing biological activity to be directly applied to livestock production and clinical research.

中图分类号:

S828
S815.4

王蜀金，周琳，陈惠娜，张正帆，黄艳玲，郭春华，王永. 重组猪表皮生长因子在酿酒酵母中的克隆表达及其生物学活性鉴定[J]. 畜牧兽医学报, 2014, 45(12): 1971-1980.

WANG Shu-jin，ZHOU Lin，CHEN Hui-na，ZHANG Zheng-fan，HUANG Yan-ling，GUO Chun-hua,WANG Yong. Cloning and Expression of Recombinant Pig Epidermal Growth Factor in Saccharomyces cerevisiae and Its Biological Activity[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(12): 1971-1980.

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重组猪表皮生长因子在酿酒酵母中的克隆表达及其生物学活性鉴定

Cloning and Expression of Recombinant Pig Epidermal Growth Factor in Saccharomyces cerevisiae and Its Biological Activity

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