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pNZ8149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pNZ8149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pNZ8149-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pNZ8149-Tα1-cIFN-γ-HA/NZ3900(rL.lactis-Tα1-cIFN-γ)并经PCR和测序鉴定;将重组乳酸乳球菌rL.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,rL.lactis-Tα1-cIFN-γ经PCR扩增到3 185 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31 μg/mL.以重组乳酸乳球菌裂解液(含重组蛋白40 μg/mL,重组菌2×109 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ConA+Ionomycin、ConA+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软件等对测序结果进行beta多样性、alpha多样性、菌群结构组成以及菌种重要性的LEfSe分析.beta及alpha多样性分析结果显示,实验组和阴性对照组鸡盲肠粪便样品测序结果分散于不同象限;且两组间样品的alpha多样性存在明显差异,表明实验组鸡具有更大的菌种丰度.菌群结构和组成分析结果显示,口服rL.lactis-Tα1-cIFN-γ显著改变了鸡盲肠肠道菌群门水平和属水平的结构和组成,其中厚壁菌门的乳酸杆菌属、普拉梭菌属和黏液真杆菌属等有益菌群显著增加;而拟杆菌门中的瘤胃菌属、另枝菌属等机会致病菌属显著减少.菌种重要性的LEfSe分析结果显示,与阴性对照组相比,口服rL.lactis-Tα1-cIFN-γ对SPF鸡肠道菌群的组成有显著影响,来自杆菌纲、乳杆菌目、乳杆菌科、乳杆菌属和厚壁菌门等有益菌成为肠道菌群内的重要菌种.上述结果首次表明,口服rL.lactis-Tα1-cIFN-γ不仅显著提高鸡PBMC的增殖活性等细胞免疫反应,而且还可以改善肠道菌群结构和组成,提高有益菌属的丰度,降低机会致病菌的丰度.本研究为天然绿色新型口服免疫增强剂和免疫佐剂的研制奠定了基础.
In order to construct recombinant Lactococcus lactis expressing thymus peptide and chicken interferon-γ fusion 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-γ gene with a G4S-linker was used as the template to amplify the Tα1-cIFN-γ gene 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-γ was 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-γ protein,and the standard curve was determined by the BCA protein assay kit.The absolute quantification of the expression of Tα1-cIFN-γ showed 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,2×109 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,respectively.The results showed that the proliferative activity of PBMC in the experimental group was significantly higher than that in the negative control group(chickens with oral PBS)at 2 weeks,4 weeks,and 7 weeks after the first immunization after stimulation(P<0.001,P<0.0001,P<0.0001).Seven weeks later,the cecal fecal samples of each group of chickens were dissected and amplified by PCR to amplify the V3-V4 gene sequence in the conserved region of the 16S rDNA gene.Analysis of LEfSe for strain importance,beta diversity,alpha diversity,and microbiota structure was carried out by high-throughput sequencing and SIMCA software,MOTHUR program,and AMDIS software.The results of beta diversity and alpha diversity analysis showed that the sequencing results of chicken cecal fecal samples in the experimental and negative control groups were scattered in different quadrants,and significant differences in alpha diversity between the two groups,indicating that the experiment group of chickens had a greater abundance of strains.The results of microbiota structure and composition analysis showed that oral administration of r L.lactis-Tα1-cIFN-γ significantly changed the structure and composition of intestinal microbiota in cecal feces,among which the beneficial microflora of Lactobacillus,Faecalibacterium prausnitzii and Blautia in Firmicutes increased significantly,while the opportunistic pathogens,such as Alistipes and the Rumen spp.,were reduced considerably.The LEfSe analysis of the importance of bacterial species showed that oral rL.lactis-Tα1-cIFN-γ had a significant effect on the composition of the intestinal microbiota of SPF chickens,and beneficial bacteria from Bacillus,Lactobacillus,Lactobacillus,Lactobacillus,and Firmicutes became essential species in the intestinal microbiota.These results are the first to suggest that oral r L.lactis-Tα1-cIFN-γ not only significantly improves the proliferative activity and cellular immune responses of chicken PBMCs,but also improves the structure and composition of intestinal flora,increases the abundance of beneficial bacteria,and reduces the abundance of opportunistic pathogens.This study lays a foundation for developing novel natural green oral immune enhancers and immune adjuvants.