重组GCRV-VP6枯草杆菌芽孢口服免疫对草鱼肝肠功能影响

Effects of orally administrated Bacillus subtilis spores expressing VP6 protein from GCRV on liver and intestine of grass carp

目的检测重组表达草鱼呼肠孤病毒(GCRV)结构蛋白VP6(GCRV-VP6)的枯草杆菌芽孢(Bacillus subtilis)口服免疫对草鱼肝肠功能影响,以初步评价其作为草鱼出血病候选疫苗的安全性。方法将前期研究获得的衣壳表达GCRV-VP6的重组枯草杆菌(B.s-CotC-GCRV-VP6)诱导形成芽孢连续口服免疫草鱼6周,在第8周搜集样品,HE染色检测B.s-CotC-GCRV-VP6芽孢口服免疫对肠道组织的病理改变及肠绒毛的影响;扫描电镜观察其对草鱼后肠表面微绒毛及黏膜表面分泌的影响;qRT-PCR检测其对肠道组织紧密连接蛋白(Claudin b、Claudin 3、Claudin 12、Claudin C、ZO-1、Occludin)转录水平的影响;HE染色检测草鱼肝组织病理改变,微板法检测血清转氨酶(AST/GOT,ALT/GPT)活性。结果HE染色结果显示口服免疫B.s-CotC-GCRV-VP6芽孢后草鱼肠道无坏死,无炎症细胞浸润等病理改变,可促进肠绒毛的生长(与对照组比较P<0.05);电镜扫描也显示口服重组芽孢可促进肠道黏膜表面微绒毛的生长和分泌孔的增加;q RT-PCR结果显示6种紧密连接蛋白在B.s-CotC-GCRV-VP6组草鱼肠道组织中转录增加,且差异有统计学意义(P<0.05);HE染色显示,重组芽孢免疫组后草鱼肝组织未见病理性改变;实验组血清转氨酶活性相对降低。结论重组B.s-CotC-GCRV-VP6芽孢口服免疫草鱼后,其肝肠不产生明显的病理性改变,对肝功能未见明显影响,并可促进其肠道绒毛和微绒毛生长及紧密连接蛋白的表达,可能有利于其肠道的消化吸收和黏膜屏障功能的提高,提示该重组芽孢作为预防草鱼出血病候选口服疫苗对肝肠无副作用。

Objective To investigate the effects of orally administrated recombinant Bacillus subtilis(B.subtilis)spores expression capsid protein VP6 from grass carp reovirus(GCRV)on liver and intestinal of grass carp and evaluate its potential as a vaccine candidate.Methods B.s-CotC-GCRV-VP6 strain was induced to be sporulated,and then fed to grass carp at 1010/g forage.Samples were collected at week 8.The intestinal tissues were stained by HE to observed pathological change of intestinal villi.The microvilli and mucosal surface secretion of grass carp hindgut were detected by scanning electron microscopy.In addition,the transcription levels of tight junction proteins(Claudin b,Claudin 3,Claudin 12,Claudin C,ZO-1,and Occludin)in the intestinal tissue were measured by qRT-PCR.Liver tissue was stained with HE and serum transaminase(AST/GOT,ALT/GPT)were detected by microplate method.Results The intestinal HE staining showed that no inflammation and necrosis were observed in the intestine tissues from fish with oral treatment of recombinant B.s-CotC-GCRV-VP6 spores.B.s-CotC-GCRV-VP6 spores could promote the growth of intestinal villi with statistically significant(P<0.05).Electron microscopy results also displayed that B.s-CotC-GCRV-VP6 spores promoted the growth of microvilli on the surface of the intestinal mucosa and increased secretory pores.Furthermore,the increases in mRNA levels for six tight junction proteins of grass carp were observed with statistically significant(P<0.05)in B.s-CotC-GCRV-VP6 group.In B.s-CotC-GCRV-VP6 treated grass carp,pathological changes were hardly found in liver tissue and the lower serum transaminase activity was observed.Conclusions There was no side-effect on the liver and intestines of grass carp orally administrated with B.s-CotC-GCRV-VP6 spores.B.s-CotC-GCRV-VP6 spores could promote the growth of intestinal villi and microvilli and increased the transcription of tight junction proteins,which is beneficial to the digestive absorption of the intestine and strengthens the mucosal barrier function.In addition,B.s-CotC-GCRV-VP6 spores have no significant side-effect on liver function.