表面展示柔嫩艾美耳球虫EtMIC2蛋白的侵入型乳酸菌的免疫特性研究
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摘要
为了研究表达pgsA′-EtMIC2融合蛋白的侵入型乳酸菌的免疫特性,将pgsA′-EtMIC2基因片段插入到载体pSIP409-FnBPA中,得到pSIP409-FnBPA-pgsA′-EtMIC2重组质粒,并用电转化的方式转入植物乳杆菌NC8感受态细胞中,构建表面展示EtMIC2蛋白的侵入型乳酸菌。通过检测免疫雏鸡外周血淋巴细胞中CD3~+CD4~+、CD3~+CD8~+T淋巴细胞的发育情况,血清中EtMIC2抗体、IL-4、IL-18、IFN-γ的含量以及肠道灌洗液中IgA的分泌量来评价该侵入型乳酸菌对雏鸡的免疫特性。结果显示,成功构建出携带pSIP409-FnBPA-pgsA′-EtMIC2质粒的重组乳酸菌;雏鸡免疫后外周血淋巴细胞中CD3~+CD4~+、CD3~+CD8~+T淋巴细胞含量明显升高;EtMIC2抗体、IL-18、IFN-γ细胞因子及IgA产生的水平均显著上升,表明该侵入型乳酸菌具有一定的免疫效力,为后期研究抗球虫的免疫保护效果奠定了基础。
In order to study the immune properties of the invasive lactic acid bacteria expressing the pgs A'-Et MIC2,the gene fragment was inserted into the p SIP409-Fn BPA to obtain the p SIP409-Fn BPApgs A'-Et MIC2,and was transfered into Lactobacillus plantarum NC8 by electroporation to construct an invasive lactic acid bacteria displaying Et MIC2 protein of Eimeria tenella on its surface. After immunization with the recombinant bacteria,its influence on T lymphocytes was detected by flow cytometry,and the expression of Et MIC2,IL-4,IL-18,IFN-γ and s Ig A was also tested by ELISA. The result showed that recombinant lactic acid bacteria carrying the p SIP409-Fn BPA-pgs A'-Et MIC2 plasmid were constructed successfully. Compared with the control group,the contents of CD3~+CD4~+ and CD3~+CD8~+T lymphocytes were significantly higher and the expression of anti-Et MIC2,IL-18,IFN-γ in serum were increased,also s Ig A in intestinal lavage fluid. Together,the recombinant bacteria,to some degree,could provide immune effects,and laid the foundation for the evaluation of the immune protection against coccidiosis.
In order to study the immune properties of the invasive lactic acid bacteria expressing the pgs A'-Et MIC2,the gene fragment was inserted into the p SIP409-Fn BPA to obtain the p SIP409-Fn BPApgs A'-Et MIC2,and was transfered into Lactobacillus plantarum NC8 by electroporation to construct an invasive lactic acid bacteria displaying Et MIC2 protein of Eimeria tenella on its surface. After immunization with the recombinant bacteria,its influence on T lymphocytes was detected by flow cytometry,and the expression of Et MIC2,IL-4,IL-18,IFN-γ and s Ig A was also tested by ELISA. The result showed that recombinant lactic acid bacteria carrying the p SIP409-Fn BPA-pgs A'-Et MIC2 plasmid were constructed successfully. Compared with the control group,the contents of CD3~+CD4~+ and CD3~+CD8~+T lymphocytes were significantly higher and the expression of anti-Et MIC2,IL-18,IFN-γ in serum were increased,also s Ig A in intestinal lavage fluid. Together,the recombinant bacteria,to some degree,could provide immune effects,and laid the foundation for the evaluation of the immune protection against coccidiosis.
引文
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[3]REID A J,BLAKE D P,ANSARI H R,et al.Genomic analysis of the causative agents of coccidiosis in domestic chickens[J].Genome Research,2014,24(10):1676-1685.
[4]WITCOMBE D M,SMITH N C.Strategies for anti-coccidial prophylaxis[J].Parasitology,2014,141(11):1379-1389.
[5]GUNAL M,YAYLI G,KAYA O,et al.The effects of antibiotic growth promoter,probiotic or organic acid supplementation on performance,intestinal microfloraandtissueofbroilers[J].InternationalJournal of Poultry Science,2006,5(2):149-155.
[6]KOTZAMANIDISC,KOURELISA,LITOPOULOU-TZANETAKI E,et al.Evaluation of adhesion capacity,cell surface traits and immunomodulatory activity of presumptive probiotic Lactobacillus strains[J].International Journal of Food Microbiology,2010,140(2):154-163.
[7]KARCZEWSKI J,TROOST F J,KONINGS I,et al.Regulation of human epithelial tight junction proteins by Lactobacillus plantarum in vivo and protective effects on the epithelial barrier[J].American Journal of Physiology Gastrointestinal and Liver Physiology,2010,298(6):G851-G859.
[8]ISOLAURI E,S TAS Y,KANKAANP P,et al.Probiotics:effects on immunity[J].American Journal of Clinical Nutrition,2001,73(2Suppl):444s-450s.
[9]蔡若鹏.基于pgsA作为表面展示元件的重组植物乳杆菌表达系统的构建及验证[D].长春:吉林农业大学,2015.CAI Ruo-peng.Construction and validation of recombinant Lactobacillus plantarum expression system based on pgs A as surface display component[D].Changchun:Jilin Agricultural University,2015.(in Chinese)
[10]姜延龙,王春凤,刘晶.一种基于FnBPA蛋白的侵入型乳酸菌:中国,106544352 A[P].2017-03-29.JIANG Yan-long,WANG Chun-feng,LIU Jing.An invasive lactic acid bacteria based on FnBPA:China,106544352 A[P].2017-03-29.(in Chinese)
[11]刘晶,王成宇,高兴,等.表达Rck蛋白的乳酸菌的构建[J].中国兽医科学,2016,46(8):1003-1008.LIU Jing,WANG Cheng-yu,GAO Xing,et al.Construction of lactic acid bacteria expressing Rck protein[J].Chinese Veterinary Science,2016,46(8):1003-1008.(in Chinese)
[12]LOEFLER D I M L,SCHOEN CU,GDEBEL W,et al.Comparison of different live vaccine strategies in vivo for delivery of protein antigen or antigen-encoding DNA and mRNA by virulence-attenuated Listeria monocytogenes[J].Infection and Immunity,2006,74(7):3946-3957.
[13]BERMU DEZ-HUMARA魣N L G.Lactococcus lactis as a live vector for mucosal delivery of therapeutic proteins[J].Human Vaccines,2009,5(4):264-267.
[14]刘晶,杨桂连,王春凤,等.乳酸菌作为DNA疫苗载体的研究进展[J].中国兽医杂志,2016,52(7):67-69.LIU Jing,YANG Gui-lian,WANG Chun-feng,et al.Research progress of lactic acid bacteria as DNAvaccine carrier[J].Chinese Journal of Veterinary Medicine,2016,52(7):67-69.(in Chinese)
[15]LI K,GAO H L,GAO L,et al.Adjuvant effects of interleukin-18 in DNA vaccination against infectious bursal disease virus in chickens[J].Vaccine,2013,31(14):1799-1805.