摘要
本研究采用PichiaPink^(TM)毕赤酵母表达系统对牦牛胃溶菌酶进行异源表达,以探索提高重组牦牛胃溶菌酶表达量的方法以及重组牦牛胃溶菌酶对大肠杆菌O111所致小鼠腹泻的治疗作用。试验选取10只体重相近的无特定病原体昆明小鼠,用大肠杆菌O111建立小鼠腹泻模型;建模成功后取30只小鼠随机分为6组,每组5只,设对照组、腹泻模型组、牦牛胃溶菌酶组、重组牦牛胃溶菌酶组、抗生素治疗组、溶菌酶产品治疗组,除对照组和腹泻模型组外其余各组灌胃给药,记录小鼠采食量及体重;第4天采血后全部扑杀,取小鼠十二指肠组织制切片做形态学观察;取肠道内容物进行肠道菌群Alpha和Beta多样性分析。结果表明:重组牦牛胃溶菌酶在诱导96 h时表达量最高,分子质量约为15.6 ku,比活性为1428.58 U/mg。攻毒后腹泻模型组、溶菌酶产品治疗组与对照组相比采食量显著降低(P<0.05);腹泻模型组与对照组相比,白细胞数、淋巴细胞数和中性粒细胞数均有极显著升高(P<0.01);十二指肠病理解剖观察发现,牦牛胃溶菌酶组肠道绒毛有所增长且厚度增加;重组牦牛胃溶菌酶组肠绒毛有所恢复。Alpha多样性分析发现,牦牛胃溶菌酶组的Simpson指数与对照组相比显著增加(P<0.05);腹泻模型组的变形菌门和弯曲杆菌门相对丰度显著增加(P<0.05);重组牦牛胃溶菌酶组和牦牛胃溶菌酶组的厚壁菌门和拟杆菌门相对丰度有所增加。本研究表明,牦牛胃溶菌酶对大肠杆菌引起的腹泻有较好的治疗效果。由于牦牛胃溶菌酶具备较强抗胃蛋白酶能力,其原酶及重组酶在饲料添加剂以及在食品加工和医疗卫生等领域具有一定的应用潜力。
This study aimed to express yak gastric lysozyme in PichiaPinkTM Pichia pastoris expression system to explore methods to improve the expression level of recombinant yak gastric lysozyme,and to investigate the therapeutic effect of recombinant yak gastric lysozyme on diarrhea caused by Escherichia coli O111 in mice.Ten mice with similar body weight were selected,and a mouse diarrhea model was established by Escherichia coli O111.After successful modeling,30 mice were randomly divided into six groups,with five mice in each group.A control group,a diarrhea model group,a yak gastric lysozyme group,a recombinant yak gastric lysozyme group,an antibiotic treatment group,and a lysozyme product treatment group were set up,excluding the control group and the diarrhea model group,the rest of the groups were given oral administration,and the feed intake and body weight of the mice were recorded.On the fourth day,all the mice were sacrificed,and the duodenal tissue sections were prepared for morphological observation.The intestinal contents were analyzed for Alpha and Beta diversity.The results showed that the highest expression level of recombinant yak gastric lysozyme was observed at 96 h after induction.The molecular weight was about 15.6 ku,and the specific activity was 1428.58 U/mg.After challenge,the feed intake of the diarrhea model group,the lysozyme product treatment group,and the control group was significantly lower than that of the control group(P<0.05);compared with the control group,the white cell count,lymphocyte count,and neutrophil count of the diarrhea model group were significantly increased(P<0.01);the pathological observation of the duodenal tissue found that the intestinal villi of the yak gastric lysozyme group increased in length and thickness;the intestinal villi of the recombinant yak gastric lysozyme group were restored.Alpha diversity analysis showed that the Simpson index of the yak gastric lysozyme group was significantly larger than that of the control group(P<0.05);the relative abundances of Pr
作者
杨杜基
袁小迪
李飙
张雨寒
孙鸿炜
刘益丽
袁国荣
江明锋
YANG Duji;YUAN Xiaodi;LI Biao;ZHANG Yuhan;SUN Hongwei;LIU Yili;YUAN Guorong;JIANG Mingfeng(College of Animal Husbandry and Veterinary Medicine,Southwest Minzu University,Chengdu 610041,China;Maoxian Bureau of Science,Technology,Agriculture and Animal Husbandry,Maoxian 623200,China)
出处
《动物营养学报》
CAS
CSCD
北大核心
2024年第3期1952-1963,共12页
CHINESE JOURNAL OF ANIMAL NUTRITION
基金
四川省科技计划项目(2023YFQ0076)
四川省重点研发项目(2021YFN0001)
四川省科技计划项目(2021YFYZ0001)。
