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

Abstract

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.

Key words: epidermal growth factor, Saccharomyces cerevisiae, cloning and expression, biological activity

CLC Number:

S828
S815.4

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.

References

［1］PLUSKE J R，HAMPSON D J，WILLIAMS I H.Factors influencing the structure and function of the small intestine in the weaned pig：a review ［J］.Livest Prod Sci，1997，51(1)：215-236.
［2］JAEGER L A，LAMAR C H，CLINE T R，et al.Effect of orally administered epidermal growth factor on the jejunal mucosa of weaned pigs ［J］.Am J Vet Res，1990，51(3)：471-474.
［3］BARNARD J A，BEAUCHAMP R D，RUSSELL W E，et al.Epidermal growth factor-related peptides and their relevance to gastrointestinal pathophysiology ［J］.Gastroenterology，1995，108(2)：564-580.
［4］PLAYFORD R J，WRIGHT N A.Why is epidermal growth factor present in the gut lumen? ［J］.Gut，1996，38(3)：303-305.
［5］CHAO J C J，LIU K Y，CHEN S H，et al.Effect of oral epidermal growth factor on mucosal healing in rats with duodenal ulcer ［J］.World J Gastroenterol，2003，9(10)：2261-2265.
［6］MAJUMDAR A P N.Postnatal under nutrition：effect of epidermal growth factor on growth and function of the gastrointestinal tract in rats ［J］.J Pediat Gastroenterol Nutr，1984，3(4)：618-625.
［7］LEE D N，KUO T Y，CHEN M C，et al.Expression of porcine epidermal growth factor in Pichia pastoris and its biology activity in early-weaned piglets ［J］. Life Sci，2006，78(6)：649-654.
［8］CHEUNG Q C K，YUAN Z，DYCE P W，et al.Generation of epidermal growth factor-expressing Lactococcus lactis and its enhancement on intestinal development and growth of early-weaned mice ［J］.Am J Clin Nutr，2009，89(3)：871-879.
［9］OKA T，SAKAMOTO S，MIYOSHI K，et al.Synthesis and secretion of human epidermal growth factor by Escherichia coli ［J］.Proc Natl Acad Sci，1985，82(21)：7212-7216.
［10］YAMAGATA H，NAKAHAMA K，SUZUKI Y，et al.Use of Bacillus brevis for efficient synthesis and secretion of human epidermal growth factor ［J］. Proc Natl Acad Sci，1989，86(10)：3589-3593.
［11］汪洋，杨桂香，吴波浪，等.猪表皮生长因子在毕赤酵母中的表达及鉴定［J］.中国农业科学，2007，40(11)：2593-2599.
［12］PASCALL J C，JONES D S C，DOEL S M，et al.Cloning and characterization of a gene encoding pig epidermal growth factor ［J］.J Mol Endocrinol，1991，6(1)：63-70.
［13］REEVES P G，NIELSEN F H，FAHEY J G C.AIN-93 purified diets for laboratory rodents：final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet ［J］.J Nutr，1993，123(11)：1939-1951.
［14］ODLE J，ZIJLSTRA R T，DONOVAN S M.Intestinal effects of milkborne growth factors in neonates of agricultural importance ［J］. J Anim Sci，1996，74(10)：2509-2522.
［15］MITTINEN P J，PERHEENTUPA J，OTONLOSKI T，et al.EGF- and TGF-alpha-like peptides in human fetal gut ［J］.Pediat Res，1989，26(1)：25-30.
［16］KANG P，TOMS D，YIN Y，et al.Epidermal growth factor-expressing Lactococcus lactis enhances intestinal development of early-weaned pigs ［J］. J Nutr，2010，140(4)：806-811.
［17］LEE D N，CHANG W F，YU I T，et al.Effects of diets supplemented with recombinant epidermal growth factor and glutamine on gastrointestinal tract development of early-weaned piglets ［J］.Asia Austra J Anim Sci，2008，21(4)：582.
［18］HAMILTON R，WATANABE C K，DEBOER H A.Compilation and comparison of the sequence context around the AUG startcodons in Saccharomyces cerevisiae mRNAs ［J］.Nucleic Acids Res，1987，15(8)：3581-3593.
［19］KANG P，TOMS D，YIN Y L，et al.Epidermal growth F factor-expressing Lactococcus lactis enhances intestinal development of early-weaned pigs［J］.J Nutr，2010，140：806-811.
［20］TANG X S，TANG Z R，WANG S P，et al.Expression，purification，and antibacterial activity of bovine lactoferrampin-lactoferricin in Pichia pastoris［J］.Appl Biochem Biotechnol，2012，166：640-651.

[1]	JIN Xin, ZHANG Man, YANG Yinfeng. Syk is Involved in the Expression of SBD-1 in Ovine Ruminal Epithelial Cells Induced by Saccharomyces cerevisiae Mannan [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(5): 1082-1090.
[2]	JIN Xin, ZHANG Man, YANG Yinfeng. Syk is Involved in the Expression of SBD-1 in Ovine Ruminal Epithelial Cells Induced by Saccharomyces cerevisiae Mannan [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(5): 1082-1090.
[3]	WANG Yunhe, JIN Xin, ZHANG Man, WEI Fang, WEN Jingyi, ZHAO Feifei, YANG Yinfeng. Effects of Saccharomyces Cerevisiae and Its Inactivated Bacteria on the Expression of β-defensin-1 in Ruminal Explants of Sheep [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(3): 581-591.
[4]	WANG Yunhe, JIN Xin, ZHANG Man, WEI Fang, WEN Jingyi, ZHAO Feifei, YANG Yinfeng. Effects of Saccharomyces Cerevisiae and Its Inactivated Bacteria on the Expression of β-defensin-1 in Ruminal Explants of Sheep [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(3): 581-591.
[5]	JIN Xin, ZHANG Man, TIAN Qiao-zhen, LIU Jiao, WANG Yun-he, YANG Yin-feng. Preliminary Study on the Signaling Pathways of SBD-1 Expression in Sheep Rumen Epithelial Cells Induced by Saccharomyces cerevisiae [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(3): 561-569.
[6]	JIN Xin, ZHANG Man, TIAN Qiao-zhen, LIU Jiao, WANG Yun-he, YANG Yin-feng. Preliminary Study on the Signaling Pathways of SBD-1 Expression in Sheep Rumen Epithelial Cells Induced by Saccharomyces cerevisiae [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(3): 561-569.
[7]	ZHANG Juan, WANG Jun-wu, HAN Ye-chen, YUN Shi-yu, WU Hai-ping, ZHAO Dong-dong, WEN Shuang, WANG Zhuo, CHENG Jia, SU Xiao-fei, ZHU Yu-bo, SUN Yao-gui, WANG Wen-kui, LI Hong-quan, DUAN Zhi-bian. The Effect of the Compound Traditional Chinese Medicine on Endothelium Derived Multiplication Factor in Lung of Broilers with Ascites Syndrome [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(1): 174-184.
[8]	ZHANG Juan, WANG Jun-wu, HAN Ye-chen, YUN Shi-yu, WU Hai-ping, ZHAO Dong-dong, WEN Shuang, WANG Zhuo, CHENG Jia, SU Xiao-fei, ZHU Yu-bo, SUN Yao-gui, WANG Wen-kui, LI Hong-quan, DUAN Zhi-bian. The Effect of the Compound Traditional Chinese Medicine on Endothelium Derived Multiplication Factor in Lung of Broilers with Ascites Syndrome [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(1): 174-184.
[9]	ZHANG Yi-fu，PAN Yang-yang，WEN Ze-xing，YU Si-jiu. The Effect of Epidermal Growth Factor on the Expression of Hypoxia Inducible Factor-1α in Cumulus Cells of Yak (Bos grunniens) and Its Correlation Analysis with Apoptosis [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(6): 1154-1161.
[10]	ZHANG Yi-fu，PAN Yang-yang，WEN Ze-xing，YU Si-jiu. The Effect of Epidermal Growth Factor on the Expression of Hypoxia Inducible Factor-1α in Cumulus Cells of Yak (Bos grunniens) and Its Correlation Analysis with Apoptosis [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(6): 1154-1161.
[11]	WANG Shu-jin，WANG Bin-xing，GUO Chun-hua，BAI Xue，PENG Zhong-li，ZHANG Zheng-fan，ZHOU Lin. Application Research of Recombinant EGF-expressed Saccharomyces cerevisiae in Early-weaned Piglets [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(5): 944-954.
[12]	WANG Shu-jin，WANG Bin-xing，GUO Chun-hua，BAI Xue，PENG Zhong-li，ZHANG Zheng-fan，ZHOU Lin. Application Research of Recombinant EGF-expressed Saccharomyces cerevisiae in Early-weaned Piglets [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(5): 944-954.
[13]	SHAO Jun-gao，ZHANG Bao-jiang，LI Shan-chun，WANG Cai-die，WANG Guo-qin，SU Yan. Comparison and Analysis of the Biological Activity of Antiserum after Immunizing with Expressed Different Domains of Staphylococus aureus Adhesin FnBPA Isolated from Bovine Milk [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(7): 1208-1214.
[14]	SHAO Jun-gao，ZHANG Bao-jiang，LI Shan-chun，WANG Cai-die，WANG Guo-qin，SU Yan. Comparison and Analysis of the Biological Activity of Antiserum after Immunizing with Expressed Different Domains of Staphylococus aureus Adhesin FnBPA Isolated from Bovine Milk [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(7): 1208-1214.
[15]	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.

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

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments