表达牛乳铁蛋白肽的重组鸡源乳酸杆菌抗IBDV感染的研究
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摘要
为探究表达牛乳铁蛋白肽的重组鸡源乳酸杆菌pPG-XLFEC/M11对雏鸡抵抗传染性法氏囊病病毒(IBDV)感染的作用,本研究利用IBDV CEF94株感染DF-1细胞,通过CCK-8法检测病毒感染后的细胞活性,利用qRT-PCR检测病毒载量,结果显示重组乳酸菌表达的牛乳铁蛋白肽可以抑制IBDV在DF-1细胞内的增殖;以重组乳酸菌pPG-XLFEC/M11饲喂雏鸡,利用IBDV强毒UK661株进行攻毒实验,结果显示饲喂重组菌的雏鸡相比于对照组,IBDV的感染对其组织器官的损伤较小,总免疫球蛋白IgG和SIgA含量增加,细胞因子IL-6、IL-8、TNF-α和IFN-γ以及TLR2的mRNA转录水平均显著升高,且提高了雏鸡的存活率。本实验结果表明,重组鸡源乳酸杆菌表达的牛乳铁蛋白肽可以增强机体的免疫应答水平,对雏鸡具有一定的抗IBDV感染的作用。本研究为表达牛乳铁蛋白肽的重组鸡源乳酸杆菌在生产中的应用以及为禽类疾病的预防奠定基础。
In this study, we tried to determine whether a recombinant chicken origin Lactobacilli/pPG-XLFEC/M11 expressing bovine lactoferrin peptide has effect on protecting chicks from being infected by bursal disease virus(IBDV). The cell viability of DF-1 cells after infected by IBDV CEF94 strain was analyzed by CCK-8, and the viral load in the cells was detected by qRT-PCR.Results showed that the bovine lactoferrin peptide expressed by the recombinant Lactobacilli inhibited the proliferation of the virus in vitro. Chicks were challenged with IBDV virulent UK661 strain after feeding with the recombinant Lactobacilli. We observed that the recombinant Lactobacilli was more potent than other groups in attenuating the tissues and organs damage induced by IBDV virulent UK661 strain. Moreover, compared to the other groups, the recombinant Lactobacilli enhanced the expression levels of IgG and SIgA in serum, significantly increased the mR NA levels of IL-6, IL-8, TNF-α, IFN-γ and TLR2, and increased the survival rate of chicks post the challenging. These results suggested that recombinant chicken origin Lactobacilli/pPG-XLFEC/M11 expressing bovine lactoferrin peptide has a protective effect on chick infected with IBDV.
In this study, we tried to determine whether a recombinant chicken origin Lactobacilli/pPG-XLFEC/M11 expressing bovine lactoferrin peptide has effect on protecting chicks from being infected by bursal disease virus(IBDV). The cell viability of DF-1 cells after infected by IBDV CEF94 strain was analyzed by CCK-8, and the viral load in the cells was detected by qRT-PCR.Results showed that the bovine lactoferrin peptide expressed by the recombinant Lactobacilli inhibited the proliferation of the virus in vitro. Chicks were challenged with IBDV virulent UK661 strain after feeding with the recombinant Lactobacilli. We observed that the recombinant Lactobacilli was more potent than other groups in attenuating the tissues and organs damage induced by IBDV virulent UK661 strain. Moreover, compared to the other groups, the recombinant Lactobacilli enhanced the expression levels of IgG and SIgA in serum, significantly increased the mR NA levels of IL-6, IL-8, TNF-α, IFN-γ and TLR2, and increased the survival rate of chicks post the challenging. These results suggested that recombinant chicken origin Lactobacilli/pPG-XLFEC/M11 expressing bovine lactoferrin peptide has a protective effect on chick infected with IBDV.
引文
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[13]Zhang T,Xie J,Zhang M,et al.Effect of a potential probiotics Lactococcus garvieae B301 on the growth performance,immune parameters and caecum microflora of broiler chickens[J].J Anim Physiol Anim Nutr,2016,100(3):413-421.
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[15]Andersen J H,Jenssen H,Sandvik K,et al.Anti-HSV activity of lactoferrin and lactoferricin is dependent on the presence of heparan sulphate at the cell surface[J].J Med Virol,2004,74(2):262-271.
[16]Biase A M D,Pietrantoni A,Tinari A,et al.Heparin-interacting sites of bovine lactoferrin are involved in anti-adenovirus activity[J].J Med Virol,2010,69(4):495-502.
[17]詹丽娥,陆冰洋,刘华栋,等.传染性法氏囊病毒分子鉴定及序列分析[J].中国动物保健,2018,5:38-42.
[18]Cammarota M,De R M,Stellavato A,et al.In vitro evaluation of Lactobacillus plantarum DSMZ 12028 as a probiotic:emphasis on innate immunity[J].Int J Food Microbiol,2009,135(2):90-98.
[2]Baker E N,Baker H M.A Structural framework for understanding the multifunctional character of Lactoferrin[J].Biochimie,2009,91(1):3-10.
[3]Zhang Yue-lei,Lima C F,Rodrigues L R.Anticancer effects of lactoferrin:underlying mechanisms and future trends in cancer therapy[J].Nutr Rev,2014,72(12):763.
[4]唐丽杰,邢婧珉,李杰,等.猪乳铁蛋白肽LFcinP基因在巴斯德毕赤酵母中组成型表达及其抑菌活性检测[J].东北农业大学学报,2012,43(6):1-5.
[5]唐丽杰,哈卓,赵丽丽,等.猪乳铁蛋白基因的克隆及其重组乳酸菌表达系统构建[J].东北农业大学学报,2010,41(3):79-84.
[6]宋丽影,邵怡岚,李雪纯,等.表达牛乳铁蛋白肽的重组乳酸乳球菌对雏鸡抗鸡白痢沙门氏菌感染的效果研究[J].中国预防兽医学报,2017,39(3):206-209.
[7]Ingrao F,Rauw F,Lambrecht B,et al.Infectious bursal disease:A complex host-pathogen interaction[J].Dev Comp Immunol,2013,41(3):429-438.
[8]Annamalai A,Ramakrishnan S,Sachan S,et al.Prophylactic potential of resiquimod against very virulent infectious bursal disease virus(vvIBDV)challenge in the chicken[J].Vet Microbiol,2016,187:21-30.
[9]韩乐濛.组成型表达鸡致病性大肠杆菌fimA与ompC融合蛋白的重组乳酸杆菌的构建及其在鸡肠道定殖特性的研究[D].哈尔滨:东北农业大学,2016.
[10]Nandakumar M P,Shen J,Raman B,et al.Solubilization of trichloroacetic acid(TCA)precipitated microbial proteins via NaO H for two-dimensional electrophoresis[J].J Proteome Res,2003,2(1):89-93.
[11]Chen Yu-ming,Lu Zhen,Zhang Li-zhou,et al.Ribosomal protein L4 interacts with viral protein VP3 and regulates the replication of infectious bursal disease virus[J].Virus Res,2016,211:73-78.
[12]马海燕,方彧聃,张敬之.应用荧光实时定量PCR方法检测重组慢病毒滴度及其感染效率[J].生命科学研究,2009,13(5):394-398.
[13]Zhang T,Xie J,Zhang M,et al.Effect of a potential probiotics Lactococcus garvieae B301 on the growth performance,immune parameters and caecum microflora of broiler chickens[J].J Anim Physiol Anim Nutr,2016,100(3):413-421.
[14]鞠珑株.组成型乳酸菌表达载体的构建及表达效果的比较[D].哈尔滨:东北农业大学,2014.
[15]Andersen J H,Jenssen H,Sandvik K,et al.Anti-HSV activity of lactoferrin and lactoferricin is dependent on the presence of heparan sulphate at the cell surface[J].J Med Virol,2004,74(2):262-271.
[16]Biase A M D,Pietrantoni A,Tinari A,et al.Heparin-interacting sites of bovine lactoferrin are involved in anti-adenovirus activity[J].J Med Virol,2010,69(4):495-502.
[17]詹丽娥,陆冰洋,刘华栋,等.传染性法氏囊病毒分子鉴定及序列分析[J].中国动物保健,2018,5:38-42.
[18]Cammarota M,De R M,Stellavato A,et al.In vitro evaluation of Lactobacillus plantarum DSMZ 12028 as a probiotic:emphasis on innate immunity[J].Int J Food Microbiol,2009,135(2):90-98.