口服表达胸腺肽和鸡干扰素融合蛋白的重组乳酸乳球菌对鸡肠道菌群影响的研究  

Study on the effect of oral administration of recombinant Lactococcus lactis expressing thymosin and chicken interferon fusion protein on chicken gut microbiota

作　　者：刘增琪 王贺 胡洪娇 崔子扬 邱宇 张素华[1] 崔红玉[4] 王云峰 何高明[1] LIU Zeng-qi;WANG He;HU Hong-jiao;CUI Zi-yang;QIU Yu;ZHANG Su-hua;CUI Hong-yu;WANG Yun-feng;HE Gao-ming(College of Animal Science and Technology,Shihezi University,Shihezi 832002,China;Harbin Guosheng Biotechnology Co.,Ltd,Harbin 150028,China;Clinical College of Medicine,Northern University of Hebei,Zhangjiakou 075000,China;Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]石河子大学动物科技学院,新疆石河子832002 [2]哈尔滨国生生物科技股份有限公司,黑龙江哈尔滨150028 [3]河北北方大学临床医学院,河北张家口075000 [4]中国农业科学院哈尔滨兽医研究所,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2023年第10期1045-1053,共9页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点研发计划项目(2022YFD1800802、2023YFD1802600)。

摘　　要：为构建表达胸腺肽和鸡γ干扰素融合蛋白(Tα1-cIFN-γ)的重组乳酸乳球菌并检测其作为口服免疫增强剂对鸡外周血单个核细胞(PBMC)的增殖活性及鸡肠道菌群的影响,本研究将鸡胸腺肽(Tα1)和鸡干扰素(cIFN-γ)的编码基因序列(去除干扰素信号肽编码基因序列),并按照宿主菌乳酸乳球菌NZ3900株的基因组优化密码子后,以经G4S-linker连接的Tα1-cIFN-γ基因为模板,采用PCR扩增携带表达载体p NZ8149同源臂及HA标签的目的基因序列(Tα1-cIFN-γ-HA);通过PCR扩增携带Tα1-cIFN-γ-HA基因序列同源臂的全长p NZ8149载体序列,利用同源重组的方式将这两段基因连接构建重组质粒p NZ8149-Tα1-cIFN-γ-HA,并经PCR和测序鉴定正确后电转化乳酸乳球菌NZ3900感受态细胞中,构建重组乳酸乳球菌p NZ8149-Tα1-cIFN-γ-HA/NZ3900(r L.lactis-Tα1-cIFN-γ)并经PCR和测序鉴定;将重组乳酸乳球菌r L.lactis-Tα1-cIFN-γ经乳酸链球菌素(Nisin)诱导6 h~7 h,离心超声破碎后取上清采用western blot鉴定Tα1-cIFN-γ的表达。结果显示,r L.lactis-Tα1-cIFN-γ经PCR扩增到3185 bp的目的基因片段,测序结果显示目的片段完全正确;且在约25 ku处出现特异性条带,与目的蛋白Tα1-cIFN-γ大小相符,利用BCA蛋白测定试剂盒构建标准曲线,对Tα1-cIFN-γ绝对定量显示其含量为31μg/m L。以重组乳酸乳球菌裂解液(含重组蛋白40μg/m L,重组菌2×10^(9)cfu/mL)经口服免疫21日龄SPF鸡,并于28日龄加强免疫一次,于首免后2周、4周、7周采血分离PBMC,并分别采用Con A+Ionomycin、Con A+PMA及LPS刺激后,采用CCK-8法检测鸡PBMC的增殖活性。结果显示,经刺激后的各实验组鸡于首免后2周、4周、7周PBMC的增殖活性均极显著高于阴性对照组(口服PBS的鸡)(P<0.001、P<0.0001、P<0.0001);7周后剖杀各组鸡取其盲肠粪便样品经PCR扩增16S rDNA基因的保守区V3~V4基因序列,采用高通量测序并采用SIMCA软件、MOTHUR程序、AMDIS软件等�In order to construct recombinant Lactococcus lactis expressing thymus peptide and chicken interferon-γfusion protein(Tα1-cIFN-γ)and to detect its effect as an oral immune enhancer on the proliferative activity of chicken peripheral blood mononuclear cells(PBMCs)and chicken intestinal microbiota.In this study,the coding gene sequences of chicken thymus peptide(Tα1)and chicken interferon-γ(cIFN-γ)with deletion of the signal peptide sequence were codon-optimized according to the genome of the host strain,Lactococcus lactis strain NZ3900.The Tα1-cIFN-γgene with a G4S-linker was used as the template to amplify the Tα1-cIFN-γgene by PCR.The target gene sequence(Tα1-cIFN-γ-HA)carrying the homology arm of pNZ8149 was amplified by PCR.The pNZ8149 vector sequence carrying the homology arm of the Tα1-cIFN-γ-HA gene sequence was amplified by PCR,and the two segments of the gene were linked to construct a recombinant plasmid,pNZ8149-Tα1-cIFN-γ-HA,by homologous recombination.The plasmid was identified correctly by PCR and sequencing after electro-transformation of Lactococcus lactis NZ3900 receptor cells.The expression of Tα1-cIFN-γwas detected by lactic acid streptococcin(Nisin)inducing for 6-7 hours,and the supernatant was taken after centrifugal ultrasonic crushing.The results showed that a specific band appeared at 24ku,which was consistent with the size of the target Tα1-cIFN-γprotein,and the standard curve was determined by the BCA protein assay kit.The absolute quantification of the expression of Tα1-cIFN-γshowed that its content was 31μg/mL.21-week-old SPF chickens were immunized orally with recombinant Lactococcus lactis lysate(containing 40μg/mL of recombinant protein,2×10^(9) cfu/mL)of recombinant lactis bacteria and boosted once at 28 weeks of age.PBMCs were separated from blood collection at 2 weeks,4 weeks,and 7 weeks after the first immunization.The proliferative activity of PBMCs in chickens was detected by the CCK-8 method after stimulation with ConA+Ionomycin,ConA+PMA,and LPS,respect

关 键 词：胸腺肽 鸡干扰素 重组乳酸乳球菌 16S rDNA 肠道菌群 

分 类 号：S852.4[农业科学—基础兽医学]