Preparation Of ETEC K99-SIgA Complex And Its Enhancement Of Intestinal Mucosal Immune Response

Enterotoxigenic Escherichia coli(ETEC)is one of the important responsible pathogens causeing newborn piglets and weaned piglets the high morbidity and mortality,in which ETEC K99 infection cases are the most in newborn piglets.Mucosal immunity plays an important role in protecting piglets from pathogenic bacteria ETEC and maintaining intestinal homeostasis.As the core immunoglobulin of mucosal immunity,SIgA has the function of impeding the adhesion of pathogenic microorganisms and immune clearance,especially for intestinal immunity.The immune complex can effectively enhance the immune response.Therefore,in this study,we prepared the ETEC K99-SIgA immune complexes in vitro to explore whether immune complexes enhanced the intestinal mucosal immunity in order to more comprehensively understand the role of SIgA on mucosal immune response.It will lay a theoretical foundation for the design of oral immune complex vaccine.In this study,the prokaryotic expression vector of pET-32a-K99/BL21 was constructed and induced by 1.2 m M IPTG for 6 h.Then the recombinant protein K99 was purified by Ni-NTA agarose affinity chromatography.The polyclonal antibody against K99 was prepared by intraperitoneal injection of K99 protein to BALB/c mice.BALB/c mice were infected with ETEC K99.The fecal supernatant containing specific SIgA antibody was obtained.The ECEC K99-SIgA complex was prepared by His pull-down.The K99-SIgA complex was reduced by reducing agent β-mercaptoethanol and SDS,and then was identified by Western blot.The results showed that the recombinant protein K99 was highly expressed in E.coli BL21,and the purified protein at a concentration of about 4.5 mg/m L,suggesting the anti-K99 polyantibody could effectively react with ETEC K99.After the mouse orally infected with the strain ETEC K99,the specific antibody level reached the peak at 42 days after infection.The ETEC K99-SIgA complex was successfully prepared with a concentration of about 2 mg/m L to provided the materials for follow-up immunological experiments.In order to explore the effect of ETEC K99-SIgA immune complex on mucosal immune response,firstly,the small loop test was used to detect which intestinal epithelial cell ingested the K99-SIgA immune complex.Secondly,the culture model of M cells in vitro was established to further reveal the uptake of K99-SIgA immune complexes by M cells.At last,the K99-SIgA complex stimulated the DC cells to elucidate their effects on DC maturation.The spleen lymphocytes of mice infected with ETEC K99 were stimulated by ETEC K99-SIgA complex.The cytokines were detected by ELISPOT.The proliferation of spleen T lymphocytes and the CTLL-2 activity of effector T cells were detected by CCK-8 method.Small intestinal loop test showed that the K99-SIgA immune complex was mainly ingested by M cells in Payer”s Patch.The inhibitors blocked the corresponding uptake pathway were used to detect the ability uptaking the ETEC K99-SIgA complex by M cell of culture model in vitro.The results showed that the K99-SIgA complex was mainly ingested by M cells in a grid protein mediated manner.The K99-SIgA complex effectively promoted DC cell maturation to enhance its antigen presentation ability.Compared to K99 antigen alone,the ETEC K99-SIgA complex up-regulated the secretion of TNF-α,IFN-γ,IL-2,IL-4,IL-6 and IL-5 cytokines more effectively,and promoted the proliferation and activation of splenic T lymphocytes and effector T cell CTLL-2 in mice,indicating that the ETEC K99-SIgA immune complex effectively enhanced the mucosal immune response.This study provides a reference basis for the development and application of oral immune complex vaccine.