Construction Of Recombinant Lactobacillus Expressing Hemagglutinin Of H5N1and Evaluation Of Immune Response

To develop a safe, effective, and convenient vaccine for the prevention of highly pathogenic avian influenza (HPAI), we separated a Lactobacillus DLD17with a strong acid capacity and better epithelial cell adhesion ability from a healthy adult chicken cecal mucosa. By using the methods of molecular biology, we have successfully constructed two recombinant expression plasmids pMG36EH (the expressing plasmid of Lactococcus lactis) and pLEM415-pH (the expressing plasmid of Lactobacillus). After transform recombinant plasmids into lactic acid bacteria Lactococcus lactis1363and Lactobacillus acidophilus4356by electroporation, we have constructed three recombinant bacterials (LL36EH, DLD17-pH and4356-pH) with stably expressing the foreign protein hemagglutininl (HA1) of HPAI virus. Immunizations were administered orally to the BALB/c mice three times at2-week intervals to evaluate the oral immune effect. Results showed that the humoral and cellular immune responses triggered by recombinant lactobacillus (DLD17-pH) were significant higher than that in other two groups. So we selected the recombinant lactobacillus (DLD17-pH) to proceed the next orally immunizations on chicken to evaluate the mucosal and systemic immune effective. The results showed that the recombinant chicken Lactobacillus (DLD17-pH) could stably colonizationand reproduction in the cecum of chickens. It was also demonstrated that the recombinant Lactobacillus (DLD17-pH) not only could triggered the production of the specific anti-HAl IgA antibody level in the mucosa of respiratory and intestinal, but also could increase the anti-HAl IgG secreation in system level. Our research also proved that DLD17-pH could activate the cellular immunity and innate immune response to enhance the immune defense capability. Therefore, DLD17-pH could be a promising oral vaccine candidate against HPAI. The development of recombinant Lactobacillus (DLD17-pH) oral vaccine not only provides a new research idea for the prevention and control of the spread of avian flu, but also laid the foundation for the application of host lactobacillus as live vector delivery system. I will discuss it from the following the four aspects.1. Isolation, identification and screening of lactic acid bacteria in chickenFirstly,22Gram positive strain, harvested from the cecal mucosa of health adult chicken, were identified by amplified16s-rRNA fragments of each strain of bacteria, and compared with determining strains of species and strains.Combined with the results of biochemical fermentation strains, the five species of lactic acid bacteria strains in chicken were determine as the receptor into strains. In addition, five strains of acid tolerance of Lactobacillus from chicken, Caco-2cell adhesion, and antibiotic sensitivity were determined. To determine the optimal shock transformation of lactic acid bacteria transformation program, lactococcus vector plasmid plem415blank pmg36e and Lactobacillus vector plasmid transfer lactococcus1363and4356as Lactobacillus acidophilus transformation receptor positive control strain of electric shock, competent preparation and electron conditions of trial and error. After the optimal conversion options, the five strains of Lactobacillus from chicken to do transformation filter test of electric shock, the filter can accept of exogenous plasmid of Lactobacillus from chicken. These results suggestion that isolate and filter down to five strains of strain, a milk Neisseria gonorrhoeae strains of fecal gut DLD-1, a strain of Lactobacillus fermentation (DLD-5), three for salivary Lactobacillus DLD-9, DLD-13, DLD-17, respectively. Through electric shocks into screening test, we get a strain of Lactobacillus from chicken suitable for exogenous plasmid, the salivary Lactobacillus DLD-17.2. Construction of recombinant H5N1haemagglutinin expression of lactic acid bacteria and its verificationIn this chapter, we firstly designed two primer HAF1and HAF2containing restrictive enzyme SalⅠ and HindⅢ. The part of fragment HA1in H5N1Asia type high pathogenic sexual avian influenza (AIV) blood coagulation were cloned into pmd19-T vector. After measuring sequence validation,. Linearized vector pmg36e and purpose fragment HA1with restrictive enzyme SalⅠ and HindⅢ were transfer recombinant lactic acid milk Neisseria gonorrhoeae, named1363-EH, through electric shock transformation. SDS-page and Western blot results indicate that blood coagulation pigment protein in lactic acid milk Neisseria gonorrhoeae were correctly express. As the above, we also constructed the expression plasmid of recombinant hemagglutinin Lactobacillus pLEM415-pH. To clone the promoter p of lactic acid dehydrogenation enzyme (LDH), two primer P1and P2were designed. Then promoter p and blood coagulation pigment gene HA1were connected to Bacillus vector pLEM415, constructing of recombinant lactic acid Bacillus expression pLEM415-pH. Secondly, vector pLEM415-pH were transfer through the method of transformation of electric shock, obtained two recombinant hemagglutinin gene of strains of Lactobacillus, namely4356-pH and DLD17-pH. Protein electrophoresis and Western-blot were shown that the haemagglutinin of HA1were expressed in two recombinant Lactobacillus strains. In short, we have three strains of lactic acid bacteria for recombinant expression of hemagglutinin of avian influenza, namely restructuring lactococcus1363-EH, recombinant of Lactobacillus acidophilus4356-pH and DLD17-pH, which laid a solid foundation for following expression of hemagglutinin of avian influenza research of recombinant Lactobacillus oral vaccine.3. Immunity levels of mouse after immunized orally with three recombinant lactic acid bacteria expressing H5Observe antibody changes of mouse respiratory and gastrointestinal tract and whole body after oral immunized with three recombinant lactic acid bacteria1363-EH, DLD7-pH and LA4356-pH. Isolate spleen lymphocytes of immunized mouse and analyze the ratio ofCD4+/CD8+, CD19+lymphocyte subpopulations. Observe proliferation and secretion level of IFN-y and IL-4lymphocytes in vitro under the stimulus of specific antigen HAL Results indicated that IgA level was significantly increased in gastrointestinal tract after oral immunizing with this three bacteria; IgA level oral immunized with LL36EH and4356-pH was decreased with the lengthening of immunized time, while IgA level of mouse immunized with DLD-pH keep stability. These show that these three bacteria can induce local mucosal immune responses. In trachea, only chicken Lactobacillus DLD17-pH group show significant specific IgA secretion, a week later than the gut. We found that IgG antibody level and HI titers in the serum mice immunes with the three recombinant lactic acid bacteria are low, immunized, it is up to4.5(the Log2) of mice immune with specific DLD17-pH. Integrated with lymphocytes proliferation test and the ratio of CD4+/CD8+,CD19+lymphocyte subpopulations, we found that recombinant lactic acid bacteria can activate T lymphocytes and B lymphocytes in the body; which is mainly caused by the proliferation of Th2type the helper T cells, whereas no significant differences between the three bacteria. To some extent, three recombinant lactic acid bacteria which can express the avian influenza hem agglutinin can stimulate specifically humeral and cellular immune response, but immune effect different because of the strain source, characteristics. DLD17-pH did best in inducing humeral and cellular immune response, particularly it can effectively induce intestinal secretion of specific IgA antibodies in the respiratory mucosa. Herein, DLD17-pH is most suitable for mucosal immunity applied to the bird flu in poultry.4. Study on Immune effect of recombinant chicken Lactobacillus DLD17-pH in chicks immunized orallyImmune chicks orally with restructured chicken Lactobacillus DLD17-pH to research local and systemic antibody level changes in respiratory and digestive tract in chicks. Detect the colonization of DLD17-pH in chick intestinal and compare immune effects with avian influenza inactivated vaccine. The results showed that DLD17-pH can not induce the production of specific IgA in the jejunum, cecal mucosa local and trachea, but also can induce specific low-level IgG antibodies. Compared with the subcutaneous immunization of avian influenza inactivated vaccine group, DLD17-pH can significantly increase gene expression of cytokines IFN-y in the digestive and respiratory tract organizations locally, TLR-2, and AvBD, While decrease gene expression of cytokines IL-4. Increasing of DLD17-pH in the cecum shows DLD17-pH can colonize in the cecal mucosa. It keeps relatively stable on21th day. In summary, oral administration of recombinant Lactobacillus DLD17-pH can not only activate early cytokine secretion in the digestive tract local tissue, respiratory, but also can induce specific antibodies of the avian flu in whole body. Therefore, it provides a new train of thought for prevents poultry bird flu and immunity by oral immunization recombinant Lactobacillus DLD17-pH.