猪白细胞介素-2和融合抗菌肽重组酵母菌构建及其对小鼠免疫和生长的协同效应
Synergistic Effect of Recombinant Yeast Co-Expressing Porcine Interleukin-2 and Fusion Antimicrobial Peptide Gene on Immunity and Growth of Mice

中文摘要：为了开发经济实用的新型免疫调节剂，以2A自剪切技术构建重组毕赤酵母 Pichiapastoris共同表达猪白细胞介素-2（IL-2）和融合抗菌肽基因，然后以重组基因工程酵母菌饲喂ICR小鼠，以实时荧光定量PCR、ELISA和流式细胞计数评估其对小鼠免疫力和生长的影响。实验结果显示，处理组（融合抗菌肽重组酵母菌、IL-2和融合抗菌肽重组酵母菌）小鼠的生长明显优于对照组（空质粒酵母菌）（ P<0.05），且血清IgG、IgG1和IgG2a含量显著升高（ P<0.05）；TLR1、TLR4、TLR6、TLR9、IL-7、IL-15、IL-23、CD62L、IL-2、IL-4、IL-6、IL-12、CRP4和CAMP基因的表达水平显著增加（ P<0.05）。同时，处理组小鼠血液白细胞Th和Tc细胞明显增多（ P<0.05），免疫力和存活率均明显高于对照组（ P<0.05）。重组酵母菌能有效增强小鼠的先天和获得性免疫力，促进生长，可进一步开发成安全有效的免疫调节生物制剂，改善动物的生长和免疫力。
英文摘要：To develop a novel and practicable immunomodulator, the recombinant Pichiapastoris to co-express porcine interleukin-2 and fusion antimicrobial peptide gene were constructed by 2A self-cleavage technique, and then fermented to feed ICR mice by gavage to evaluate its effects on the immunity and growth of mice via real time fluorescent quantitative PCR, ELISA and flow-cytometry. It was found that the expressed molecules of recombinant yeasts not only showed remarkable immunological bioactivity but also manifested obvious antimicrobial activity in vitro. The feeding of the yeast relatively improved the growth of mice in comparison to the control group ( P<0.05). The contents of IgG, IgG1 and IgG2a were significantly increased in the yeast-treated groups ( P<0.05), and the expression levels of the TLR1, TLR4, TLR6, TLR9, IL-7, IL-15, IL-23, CD62L, IL-2, IL-4, IL-6, IL-12, CRP4 and CAMP genes significantly increased in comparison to the control ( P<0.05). Similarly, significant increases of the leukocytes, Th and Tc cells were detected in the blood of yeast-treated mice ( P<0.05). Meanwhile, the mice of yeast-treated groups displayed markedly higher levels of immunity and survival rates than those of the control after challenged with virulent bacteria Escherichia coli and Staphylococcus aureus ( P<0.05). These results suggest that the recombinant yeast can effectively enhance the immunity and growth of mice, and thus can be further developed as a promising safe and effective immunomodulator for livestock. 2018,37(6): 609-619 收稿日期：2017-11-16 分类号：Q78;Q95-33 基金项目：四川省科技项目（2016RZ0034，2017HH0023）；四川省国际科技合作项目（2010HH0003）；成都市科技攻关项目（2015NY-02-0028-NC）；四川省重点动物育种项目（2016NYZ0042）；科技部国际合作项目（2011DFA10101103） 作者简介：胡冰,女,硕士研究生,主要从事新型抗感染免疫分子制剂方面的研究,E-mail:578077613@qq.com;吴雪颖,女,硕士,医学遗传与分子生物学;马常俊,女,硕士,医学遗传与分子生物学 *通信作者：吕学斌,王泽洲,主要从事动物表观遗传学、动物分子免疫、疫苗研制、免疫应答调节和新型抗感染免疫分子制剂的研究,E-mail:lake96@qq.com;高荣,主要从事动物表观遗传学、动物分子免疫、疫苗研制、免疫应答调节和新型抗感染免疫分子制剂的研究,E-mail:gaorong96@163.com 参考文献： Bals R, Wilson JM. 2003. Cathelicidins-a family of multifunctional antimicrobial peptides[J]. Cellular and Molecular