摘要
本试验旨在构建表达猪表皮生长因子(pEGF)的重组乳酸乳球菌(L.lactis),并评价其对葡聚糖硫酸钠(DSS)诱导的结肠炎模型小鼠肠道损伤的修复作用,为将表达pEGF重组L.lactis应用于断奶仔猪肠道保护提供重要的依据。设计含有3个拷贝数的pEGF同向串联基因序列,并进行L.lactis密码子优化,将合成的3pEGF基因片段与表达载体pNZ8148连接,构建表达pEGF的重组L.lactis。选用32只BALB/c小鼠,随机分为4组,每组8只。采用4%的DSS建立小鼠结肠炎模型,各组小鼠具体处理如下:正常对照组,试验第1~12天饮用自来水;DSS模型对照组,试验第1~7天饮用4%的DSS水溶液,第8~12天饮用自来水;L.lactis组,饮水按照DSS模型对照组处理,同时每天口服1×1012 CFU L.lactis,连续12 d;重组L.lactis组,饮水按照DSS模型对照组处理,同时口服1×1012 CFU表达pEGF的重组L.lactis,连续服12 d。结果显示:1)通过序列鉴定可知,3pEGF基因成功转入L.lactis;Western blot分析表明所表达的pEGF能与特异性抗体相结合,初步证明该重组蛋白具有活性。2)与正常对照组相比,DSS模型对照组小鼠结肠长度显著降低(P<0.05);与DSS模型对照组相比,重组L.lactis组小鼠结肠长度显著增加(P<0.05)。3)与正常对照组相比,DSS模型对照组小鼠结肠闭锁蛋白(occludin)浓度显著降低(P<0.05),D-乳酸(D⁃LAC)浓度显著升高(P<0.05),二胺氧化酶(DAO)活性极显著升高(P<0.01);与DSS模型对照组相比,重组L.lactis组小鼠结肠occludin浓度显著升高(P<0.05),D⁃LAC浓度显著降低(P<0.05),DAO活性极显著降低(P<0.01)。4)与正常对照组相比,DSS模型对照组小鼠结肠白细胞介素-10(IL⁃10)浓度极显著降低(P<0.01),白细胞介素-4(IL⁃4)浓度显著降低(P<0.05),肿瘤坏死因子-α(TNF⁃α)浓度极显著增加(P<0.01);与DSS模型对照组相比,重组L.lacits组小鼠结肠IL⁃10浓度极显著增加(P<0.01),IL⁃4浓度显著增加(P<0.05),TNF⁃α浓度有降低的趋势。由此可见,本试验成功构建了表达pEGF的重组L.lacits,重组pEGF具有良好生物活性;小鼠口服表达pEGF的重组L.lacits可抑制结肠的缩短,改善结肠结构和通透性,调节细胞因子浓度到达正常水平,这说明表达pEGF的重组L.lacits维护了肠道屏障功能的完整性,对损伤的肠道具有一定修复作用。
This study aimed to construct recombinant Lactococcus lactis(L.lactis)expressing porcine epider⁃mal growth factor(pEGF),evaluate its repair effects on intestinal damage of colitis model induced by dextra sulfate sodium(DSS)in mice,and further provide an important foundation in order that recombinant L.Lactis could be applied to intestinal protection in weaned piglets.Three copies of mature pEGF with the same nucleo⁃tide sequence were designed by tandem repeat strategy.The gene sequence of designed 3pEGF was further opti⁃mized based on codon bias of L.lactis.The resulting fragment was synthesized,and then was cloned into pNZ8148 vector.Recombinant L.lactis expressing pEGF was constructed.Thirty⁃two BALB/C mice were ran⁃domly allotted in four groups with eight mice per group.Colitis model was induced by 4%DDS in mice.The mice in the four groups were treated as follow:normal control group received normal drinking water on the 1st to 12th day;DSS model control group received 4%DSS solution in drinking water on the 1st to 7th days and followed with normal drinking water on the 8th to 12th days;L.lactis group received the same treatment of DSS as DSS model control group,meanwhile,these mice were orally administered with 1×1012 CFU/d of L.lactis for consecutive 12 days;recombinant L.lactis group received the same treatment of DSS as DSS model control group,meanwhile,these mice were orally administered with 1×1012 CFU/d of recombinant L.lactis for consecutive 12 days.The results showed as follows:1)by sequence identification,the 3pEGF had been successfully transformed into L.lactis.Western blot analysis results revealed that pEGF could react with the specific antibody,which primarily indicated that pEGF had biological activity.2)DSS treatment significantly shortened colon length compared with normal control group(P<0.05),and oral administration of recombinant L.lactis significantly increased colon length compared with DSS model control group(P<0.05).3)DSS treat⁃ment significantly decreased colonic occludin concentration(P<0.05),significantly increased colonic D⁃lactate (D⁃LAC) concentration (P<0.05) and diamine oxidase (DAO) activity (P < 0.01) compared with normalcontrol group. Oral administration of recombinant L. lactis significantly increased colonic occludin concentration(P<0.05), and significantly decreased colonic D⁃LAC concentration (P<0.05) and DAO activity (P<0.01)compared with DSS model control group. 4) DSS treatment significantly decreased colonic interleukin⁃10 (IL⁃10) (P<0.01) and interleukin⁃4 (IL⁃4) concentrations (P<0.05), and significantly increased colonic tumornecrosis factor⁃α (TNF⁃α) concentration (P<0.01) compared with normal control group. Oral administrationof recombinant L. lactis significantly enhanced colonic IL⁃10 (P<0.01) and IL⁃4 concentrations (P<0.05),and significantly decreased colonic TNF⁃α concentration compared with DSS model control group. In conclu⁃sion, recombinant L. lactis expressing pEGF is successfully constructed, and recombinant pEGF possessesgood biological activity. Oral administration of recombinant L. lactis in mice can prevente colon shortening,improve intestinal permeability, and regulate the concentrations of intestinal cytokines to normal levels, theseshow that recombinant L. lactis expressing pEGF can maintain the integrity of intestinal mucosal barrier, andhas a certain effect for repairing the damaged intestinal tissue.
作者
刘淑杰
陶新
邓波
门小明
徐子伟
LIU Shujie;TAO Xin;DENG Bo;MEN Xiaoming;XU Ziwei(Institute of Animal Husbandry and Veterinary Science,Zhejiang Academy of Agricultural Sciences,Hangzhou 310021,China)
出处
《动物营养学报》
CAS
CSCD
北大核心
2021年第4期2253-2262,共10页
CHINESE JOURNAL OF ANIMAL NUTRITION
基金
浙江省重点研发计划项目(2021C02007)
国家现代农业产业技术体系建设专项资金资助(CARS⁃36)
国家青年科学基金项目(C170105/31501966)
浙江省科技厅资助项目(2016C02054)。
关键词
表皮生长因子
乳酸乳球菌
表达
肠道
损伤修复
结肠炎
epidermal growth factor
Lactococcus lactis
express
intestine
damage repair
colitis
