[1]李中圣*,伍建敏,王凤求,等.猪圆环病毒 2 型 Cap 蛋白毕赤酵母分泌表达及发酵条件优化[J].中国预防兽医学报,2017,(09):697-701.[doi:10.3969/j.issn.1008-0425.2017.09.02]
 LI Zhong-sheng*,WU Jian-min,WANG Feng-qiu,et al.Secretory expressing of recombinant PCV2 Cap protein by P.pastoris and optimization of the fermentation[J].Chinese journal of preventive veterinary medicine,2017,(09):697-701.[doi:10.3969/j.issn.1008-0425.2017.09.02]
点击复制

猪圆环病毒 2 型 Cap 蛋白毕赤酵母分泌表达及发酵条件优化
()
分享到:

《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2017年09
页码:
697-701
栏目:
病毒及其病原生物学
出版日期:
2017-09-12

文章信息/Info

Title:
Secretory expressing of recombinant PCV2 Cap protein by P.pastoris and optimization of the fermentation
文章编号:
1008-0589(2017)09-0697-05
作者:
 李中圣* 伍建敏王凤求侯月娥张杰王贵平
 (广东海大畜牧兽医研究院,广东 广州 511400)
Author(s):
 

LI Zhong-sheng* WU Jian-min WANG Feng-qiu HOU Yue-e ZHANG Jie WANG Gui-ping

 (Guangdong haid institute of animal husbandry & veterinary, Guangzhou 511400, China)
关键词:
 关键词:PCV2毕赤酵母表达发酵
Keywords:
 porcine circovirus type 2  Pichia pastoris  expressing  fermentation
分类号:
852.65
DOI:
10.3969/j.issn.1008-0425.2017.09.02
文献标志码:
A
摘要:
为实现猪圆环病毒2型(PCV2) Cap蛋白在酵母中的高效表达,本研究将去除信号肽并经序列优化后的PCV2 ORF2片段插入pPICZαA表达载体,将重组表达质粒克隆转化巴斯德毕赤酵母(Pichia pastoris) X33感受态细胞中,应用Zeocin抗性和PCR法对转化子进行初步筛选和表型鉴定,利用“双膜法”筛选出PCV2 Cap表达量相对较高的酵母。采用正交试验对优选的酵母进行发酵试验,分析培养基pH、甲醇诱导时间、甲醇诱导浓度和诱导时酵母浓度对PCV2重组Cap蛋白(rCap)分泌表达的影响。结果显示酵母分泌表达PCV2 rCap的优化条件为:培养基pH为5.0,发酵液OD600nm至1.0,加入终浓度1.0 %甲醇诱导96 h。优化后的PCV2 rCap分泌量可达207.58 μg/mL。中和试验结果显示,发酵产物PCV2 rCap免疫小鼠产生的抗体可有效中和PCV2。本研究为利用酵母表达PCV2 Cap进行疫苗研制奠定了基础。
Abstract:
To realize the high expression of porcine circovirus type 2 (PCV2) Cap protein in the yeast cells, a codon optimized PCV2 ORF2 fragment, in which the region of encoding signal peptide DNA sequence was removed, was inserted into the pPICZαA expression vector. The constructed recombinant plasmid was transformed into Pichia pastoris X33 cells. The multiple integrated transformants were preliminarily screened by Zeocin resistance, and the Mut genotypes of the selected yeast cells were identified by PCR. The potentially high expression colonies were further selected with in situ double member screening test. The fermentation test was operated with the selected yeast cell by thogonal test, and the related parameters were optimized. The optimum fermentation condition was that the medium was pH5.0, adding methanol to 1.0% when the medium OD600nm value reached to 1.0, and inducing 96 h. Under the optimum fermentation condition, the secretion of PCV2 rCap could reach 207.58 g/mL. The result of neutralization test showed that the antibody produced by PCV2 rCap immunized mice could effectively neutralize PCV2. The study laid the foundation for the vaccine development using the PCV2 rCap protein expressed by P.pastoris.

参考文献/References:

[1] Segales J, Rosell C, Domingo M. Pathological findings associated with naturally acquired porcine circovirus type 2 associated disease [J]. Vet Microbiol, 2004, 98: 137-149.
[2]Segales J. Porcine circovirus type 2 (PCV2) infections: clinical signs, pathology and laboratory diagnosis [J]. Virus Res, 2012, 164: 10-19.
[3]Blanchard P, Mahe D, Cariolet R, et al. Protection of swine against post-weaning multisystemic wasting syndrome (PMWS) by porcine circovirus type 2 (PCV2) proteins [J]. Vaccine, 2003, 21: 4565-4575.
[4]Nawagitgul P, Harms P A, Morozov I, et al. Modified indirect porcine circovirus (PCV) type 2-based and recombinant capsid protein (ORF2)-based enzyme-linked immunosorbent assays for detection of antibodies to PCV [J]. Clin Diagn Lab Immunol, 2002, 9: 33-40.
[5]Bucarey S A, Noriega J, Reyes P, et al. The optimized capsid gene of porcine circovirus type 2 expressed in yeast forms virus-like particles and elicits antibody responses in mice fed with recombinant yeast extracts [J]. Vaccine, 2009, 27: 5781- 5790.
[6]Khayat R, Brunn N, Speir J A, et al. The 2.3-angstrom structure of porcine circovirus 2 [J]. J Virol, 2011, 85: 7856-7862.
[7]Wu Pei-ching, Lin Wei-li, Wu Chi-ming, et al. Characterization of porcine circovirus type 2 (PCV2) capsid particle assembly and its application to virus-like particle vaccine development [J]. Appl Microbiol Biotechnol, 2012, 95: 1501-1507.
[8]Beach N M, Meng Xiang-jing. Efficacy and future prospects of commercially available and experimental vaccines against porcine circovirus type 2 (PCV2) [J]. Virus Res, 2012, 164;33-42.
[9]Kristensen C S, Baadsgaard N P, Toft N. A meta-analysis comparing the effect of PCV2 vaccines on average daily weight gain and mortality rate in pigs from weaning to slaughter [J]. Prev Vet Med, 2011, 98: 250-258.
[10]张晓勇,郑其升,魏雪涛,等. 猪圆环病毒II型ORF2基因在酵母中的高效分泌表达[J]. 中国病毒学, 2005,125-129.
[11]Silva J G, Coimbra E C, Jesus A L, et al. Secretory expression of Porcine Circovirus Type 2 capsid protein in Pichia pastoris [J]. J Virol Methods, 2014, 207: 226-31.
[12]Tu Ya-bin, Wang Yan-qun, Wang Gang, et al. High-level expression and immunogenicity of a porcine circovirus type 2 cap-sid protein through codon optimization in [J]. Appl Microbiol Biotechnol, 2013, 97: 2867-2875.
[13]Nainys J, Lasickiene R, Petraityte-Burneikiene R , et al. Generation in yeast of recombinant virus-like particles of porcine circovirus type 2 capsid protein and their use for a serologic assay
and development of monoclonal antibodies [J]. BMC Biotechnology, 2014, 14: 100.
[14]Zaveckas M, Snipaitis S, Pesliakas H, et al. Purification of recombinant virus-like particles of porcine circovirus type 2 capsid protein using ion-exchange monolith chromatography [J]. J Chromatogr B Analyt Technol Biomed Life Sci, 2015, 991: 21-28.
[15]Bucarey S A, Pujol M, Poblete J, et al. Chitosan microparticles loaded with yeast-derived PCV2 virus-like particles elicit antigen-specific cellular immune response in mice after oral administration [J]. Virol J, 2014, 11: 149.
[16]Patterson R, Eley T, Browne C, et al Oral application of freeze-dried yeast particles expressing the PCV2b Cap protein on their surface induce protection to subsequent PCV2b challenge
[J]. Vaccine, 2015, 33: 6199-6205.
(本文编辑:彭永刚)

相似文献/References:

[1]梁化春,齐景伟,刘淑英*,等.绵羊肺腺瘤病的病理学及RT-PCR诊断 [J].中国预防兽医学报,2009,(06):443.
 LIANG Hua-chun,QI Jing-wei,LIU Shu-ying*,et al.The pathological and RT-PCR diagnosis ofsheep pulmonary adenomatosis [J].Chinese journal of preventive veterinary medicine,2009,(09):443.
[2]刘合义,孙留霞,王进轶,等.牛冠状病毒重组N蛋白间接ELISA检测方法的建立 [J].中国预防兽医学报,2009,(08):618.
 LIU He-yi,SUN Liu-xia,WANG Jin-yi,et al.Development of an indirect ELISA for the detectionof Bovine coronavirus using recombinant N protein [J].Chinese journal of preventive veterinary medicine,2009,(09):618.
[3]秦玉寅,王雪峰,韦华冕,等.马传染性贫血病毒弱毒疫苗gp90多样性及其对病毒体外增殖的影响[J].中国预防兽医学报,2014,(03):182.[doi:10.3969/j.issn.1008-0589.2014.03.04]
 QIN Yu-yin,WANG Xue-feng,WEI Hua-mian,et al.gp90 genetic diversity of EIAV attenuated vaccine and its impact on EIAV’s proliferation in vitro[J].Chinese journal of preventive veterinary medicine,2014,(09):182.[doi:10.3969/j.issn.1008-0589.2014.03.04]
[4]刘 畅,李 磊,于立新,等.稳定表达绵羊肺腺瘤病毒受体Hyal-2蛋白细胞系的建立[J].中国预防兽医学报,2014,(03):187.[doi:10.3969/j.issn.1008-0589.2014.03.05]
 LIU Chang,LI Lei,YU Li-xin,et al. Development of a cell line stably expressing Jaagsiekte retrovirus receptor Hyal-2 protein[J].Chinese journal of preventive veterinary medicine,2014,(09):187.[doi:10.3969/j.issn.1008-0589.2014.03.05]
[5]冷青文,李志远,鲁海富,等.盘羊体内绵羊肺炎支原体的分离和鉴定[J].中国预防兽医学报,2014,(03):197.[doi:10.3969/j.issn.1008-0589.2014.03.07]
 LENG Qing-wen,LI Zhi-yuan,LU Hai-fu,et al.Isolation and identification of Mycoplasma ovipneumoniae in Argali[J].Chinese journal of preventive veterinary medicine,2014,(09):197.[doi:10.3969/j.issn.1008-0589.2014.03.07]
[6]程子英,高 洋,王 铭,等.弓形虫酵母双杂交cDNA文库构建及与AMA1羧基端相互作用蛋白的筛选[J].中国预防兽医学报,2014,(03):201.[doi:10.3969/j.issn.1008-0589.2014.03.08]
 CHENG Zi-ying,GAO Yang,WANG Ming,et al. Construction of yeast two-hybrid cDNA library of Toxoplasma gondii and screen of AMA1 c-terminal interacting proteins[J].Chinese journal of preventive veterinary medicine,2014,(09):201.[doi:10.3969/j.issn.1008-0589.2014.03.08]
[7]韩启灿,霍光华*,罗桂祥,等.野生冬菇产病原拮抗物的工艺参数确定[J].中国预防兽医学报,2014,(03):208.[doi:10.3969/j.issn.1008-0589.2014.03.10]
 HAN Qi-can,HUO Guang-hua*,LUO Gui-xiang,et al.The fermentation process of anti-pathogenic bacteria substances produced by a wild Flammulina ssp[J].Chinese journal of preventive veterinary medicine,2014,(09):208.[doi:10.3969/j.issn.1008-0589.2014.03.10]
[8]安 伟,肖 雨*,张崇文,等.鳜鱼传染性脾肾坏死病毒TaqMan荧光定量PCR检测方法的建立[J].中国预防兽医学报,2014,(03):214.[doi:10.3969/j.issn.1008-0589.2014.03.11]
 AN Wei,XIAO Yu*,ZHANG Chong-wen,et al. Establishment of a TaqMan qPCR assay for detecting the infectious spleen and kidney necrosis virus of Siniperca chuatsi[J].Chinese journal of preventive veterinary medicine,2014,(09):214.[doi:10.3969/j.issn.1008-0589.2014.03.11]
[9]胡 骑,何于雯,信爱国*,等.口蹄疫病毒实时定量RT-PCR方法的建立及初步应用[J].中国预防兽医学报,2014,(03):218.[doi:10.3969/j.issn.1008-0589.2014.03.12]
 HU Qi,HE Yu-wen,XIN Ai-guo*,et al.Development and application of a real-time RT-PCR assay for detection of foot-and-mouth disease virus[J].Chinese journal of preventive veterinary medicine,2014,(09):218.[doi:10.3969/j.issn.1008-0589.2014.03.12]
[10]周 兵,李天鹤,李 宁,等.抗鸡传染性法氏囊病病毒单链抗体库的构建及其中和抗体的筛选[J].中国预防兽医学报,2014,(03):227.[doi:10.3969/j.issn.1008-0589.2014.03.14]
 ZHOU Bing,LI Tian-he,LI Ning,et al.Construction and screening of an anti-IBDV scFv library[J].Chinese journal of preventive veterinary medicine,2014,(09):227.[doi:10.3969/j.issn.1008-0589.2014.03.14]

更新日期/Last Update: 2017-09-15