| About 30%to 50%diarrhea were caused by Enterotoxigenic Escherichia coli(Enterotoxigenic E.coli,ETEC),which caused a huge economic losses in breeding industry.ETEC planted on the intestinal surface mainly by the fimbria adhesin and then released enterotoxin which caused dehydration diarrhea for piglets.The primary factor for diarrhea caused by ETEC was the binding of the fimbria adhesin to the specific receptor of intestinal epithelial cells.F4 and F18 were the main ETEC strains which caused diarrhea of piglets.FaeG and FedF as fimbria adhesins of F4 and F18,responding,play a major role in the colonization.The fimbria adhesin mainly composed of protein and had good immunogenicity.Some studies have shown that immunizing piglets by the expression of fimbria adhesin in vitro played a great role on the prevention of diarrhea caused by pathogenic bacteria.This study was aimed at improving the soluble expression content of adhesin by constructing the co-expression system of the adhesin and chaperone GroEL/GroES.Culture conditions were optimized to add the output of the soluble expression of the adhesion for achieving the scale production of the active adhesin antigenThe immunogenicity of the recombinant adhesin protein was also assayed.The first part of results that the F4 and F18 adhesin and chaperone GroEL/GroES co-expression vector was as follows:With F4ac standard strains C8371 and F18ac standard strains 2134P as the test material,the FaeG sequences and FedF 15-165 sequences which mainly participate the process of adhesion amplified by PCR which were built on the pET expression vectors.And the Expression vectors and chaperone GroEL/GroES carrier vector pGro7 were co-transformed to the E.coli expression host BL21(DE3).According to Western-blotting detection,the co-expressed system successfully expressed target protein.By comparing the co-expressed system to the single expressed system,it was found that FaeG soluble expression level increased by 18%,inclusion body expression quantity reduced 44%,FedF soluble expression level increased by 58%,but no difference was shown between the amount of inclusion body expression.Those shown that the co-express system significantly improved FaeG and FedF soluble expression level.Adhesin protein with 6*HIS label was purified by the nickel affinity chromatography,and the adhesin protein which purity was up to 90%was gained.This provided a good antigen for subsequent immunization experiments.The result of MALDI-TOF-MS MS analysis of the purified protein showed that the consistency of amino acid sequence and natural protein sequence,FaeG and FedF respectively reached 72.5%and 69.5%.The result of circular dichroism analysis of purified protein showed that the adhesin protein conformation the correct Spatial structure.The second part of optimization the restructuring engineering bacteria fermentation soluble expression was as follows:Through a single factor optimization the culture condition of inoculum age,inducing temperature,inducting time,IPTG induced concentration and L-arabinose induced concentration,by analyzing the influence of various factors on the growth of the bacteria,combined with the Quantity one 4.6.2 software for analysis of SDS-PAGE gel band of the bacteria lysed,and calculating adhesin protein soluble expression of gray values to choose an optimal level of each factor.Optimization results which were as follows:the optimal inoculum age of BL21(FaeG)was 10h;the inducting time was 24h.the inducing temperature was 18?;the IPTG induced concentration was 0.3mmol/L;the L-arabinose induced concentration was 0.8g/L;the concentration of target protein in the culture supernatant reached 37.3mg/L,which was 8 times higher than before optimization.And the optimal inoculum age of BL21(FedF)was 10h;the induction time was 24h,the inducing temperature was 18?;the IPTG induced concentration was O.lmmol/L;the L-arabinose induced concentration was 0.8g/L;the concentration of target protein in the culture supernatant reached 12.2mg/L,but there was little target protein in the supernatant before optimization.Through single factor experiment,screening the desirable carbon sources,nitrogen source with suitable concentration and the optimal phosphate concentration.The analysis method was same to soluble fermentation condition optimization test.Finally,the optimum medium of BL21(FaeG)was composed of 10g/L glucose,18g/L yeast extract,9g/L peptone,72mmol/L potassium dihydrogen phosphate and 17mmol/L dipotassium hydrogen phosphate.The soluble expression level of FaeG protein after optimization reached 60.36mg/L,which increase 1.46 times than before(41.26mg/L).The optimum medium of BL21(FedF)was composed of 30g/L glucose,12g/L yeast extract,6g/L peptone,36mmol/L potassium dihydrogen phosphate and 8.5mmol/L dipotassium hydrogen phosphate.After optimization,the soluble expression level of FedF protein reached 21.78mg/L,which increased 1.37 times than the prior(15.9mg/L).Based on the result of shaking culture,high cell density cultivated recombinant bacteria by the way of variable speed feed in the fermenter,According to cell growth periodical fed with different concentrations of glucose,keep the medium low residual sugar concentration(<3g/L),maintain the dissolved oxygen around 30%by controlling the ventilation and rotate speeds,the final cell concentration reached more than 12g(DCW)/L,FaeG protein soluble expression level reached 536mg/L,FaeG protein soluble expression level reached 357mg/L,they were respectively nine times and fifteen times more than the shaking culture.The third part of polyclonal antibody preparation result was as belows:Immunizing male New Zealand rabbits by purified adhesin as antigen;serum was collected after four immunization;the methods of indirect ELISA was used to detect serum titer.The titer for FaeG antigen serum reached 1:320000.The titer for FedF antigen serum reached 1:40000.This study showed that two polyclonal antibodies had high antibody titer.To detect the serum antibody specificity by western blot method,it showed that the immune positive serum formed an explicit stripe at the target protein position while the negative serum did not form a stripe.this proved that both FaeG and FedF recombinant protein could obviously have a specific antigen-antibody reaction with rabbit anti-positive sera.In conclusion,the assist expression of molecular chaperone GroEL/GroES and th e optimization of fermentation conditions significantly improved the adhesin protein soluble expression levels,so the shortcomings of inclusion bodies in E.coli was overcam e to some extend.Using the F4 and F18 adhesin as immunogen for preparing a polyclon al antibody had a very high titer and immune specificity,this provided a viable alternativ e treatment way as antibiotic for the prevention of piglets diarrhea. |
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