共表达猪IL-4/6与抗菌肽融合基因的重组酵母菌的生物活性及其对小鼠免疫增强作用研究

Co-expression of porcine IL-4/6 and antimicrobial peptide fusion gene in yeast and its bioactivity

为开发高效安全经济的免疫调节剂,本研究构建了共表达猪IL-4/6和猪抗菌肽融合基因VRP的重组毕赤酵母SG46P。通过猪淋巴细胞增殖试验和抑菌试验检测其发酵上清液的生物学活性和抑菌活性,并用该重组毕赤酵母对30只21日龄雌性ICR小鼠进行灌胃接种。接种后,每周采集小鼠尾部静脉血进行免疫功能分析试验;并在第28 d用大肠杆菌和金黄色葡萄球菌进行小鼠腹腔攻毒试验。结果显示:实验组SG46P的发酵上清液较对照组能显著刺激猪淋巴细胞的增殖(p<0.05),且该发酵上清液具有明显的抑菌作用(p<0.05)。接种重组毕赤酵母SG46P的小鼠的体质量有所增加(p>0.05)且其外周血中白细胞数量、血红蛋白含量均显著高于对照组(p<0.05);其血清中Ig G、Ig G1和Ig G2a抗体水平均较对照组显著增加(p<0.05),其外周血中CD4+T和CD8+T淋巴细胞数量均显著高于对照组(p<0.05);TLR4、TLR9、IL-2、IFN-γ、IL-4、IL-6、CD62L、IL-7、IL-23、CAMP和Crp4基因的转录水平均显著高于对照组(p<0.05)。攻毒后,接种SG46P的小鼠的生存率显著高于对照组(p<0.05)。结果表明:重组毕赤酵母SG46P不仅能够显著提高小鼠免疫基因的转录水平,有效提高动物的先天和获得性免疫水平,还可明显增强小鼠抗感染能力,这为研制高效安全经济的分子免疫调节剂和防治动物传染病开拓了新途径。

To develop an efficient and economical immunomodulator, the recombinant Pichia pastoris SG46P was constructed to co-express porcine IL-4/6 and antimicrobial peptide fused gene （VRP）. The bioactivity and antibacterial activity of the expressed fusion protein in the culture medium were identified by proliferation assay of porcine lymphocytes and antibacterial assay. In addition, thirty 21-day old female ICR mice were feed with the recombinant yeasts by garage and the blood samples was collected via tail vein weekly for immunological analysis. On the 28th day, the mice were challenged by injecting E.coli and S.aureus into the abdominal cavity. The results showed that the proliferation of porcine lymphocytes was significantly promoted in the treated group （p〈0.05）, and the replication of bacteria was dramatically inhibited （p〈0.05）. After inoculation of the recombinant yeasts, the treated mice gained slightly body weight （p〉0.05）. The leukocytes and hemoglobin obviously increased in the peripheral blood of the treated mice （p〈0.05）. In comparison with the control group, the CD4＋ and CD8＋ T cells markedly raised in the blood of the treated group and the levels of IgG, IgG1 and IgG2a also significantly increased （p〈0.05）. Similarly, the expression levels of TLR4, TLR9, IL-7, IL-23, CD62L, IL-2, IL-4, IL-6, IFN-3,, CAMP and Crp4 genes were significantly higher than those in the control groups （p〈0.05）. Meanwhile, the survival rate of the treated mice was higher than that of mice in control groups after challenge with pathogenic bacteria （p〈0.05）. These results suggest that the recombinant P.pastoris SG46P could not only promote the innate and adaptive immunity in mice, but also significantly enhance the antibacterial function of mice, which could be further developed as a promising immunomodulator for the control of animal infectious diseases.