Recombinant Expression Of Swine Oral Antibodies In Pichia Pastoris And Optimization Of Induction Conditions

Antibody,secreted by organisms,is an immunoglobulin that can specifically bind with corresponding antigens.It mainly exists in the internal environment of organisms with the function of protecting their own health.In mammals,maternal antibody finds its way to the intestines of the young through breast milk,which can provide passive immunity against the infection of pathogenic microorganisms.In recent years,more and more studies have found that adding antibodies to the feed for young animals can effectively prevent them from intestinal diseases.Therefore,oral antibodies specially designed for animals are of great significance to their health.In this study,four kinds of swine oral antibodies were developed and the Pichia pastoris expression system was chosen to realize the recombinant expression.The aim is to see whether the recombinant antibodies can be expressed in Pichia pastoris,explore the influence of inducible conditions on the number of the recombinant antibodies’expressions and identify the activity of the recombinant antibodies.It is expected to provide reference for preparative technology and functional research of swine oral antibodies as well as new ideas for substitution of antibiotics.In the previously-reported studies,VHH2 and VHH3,two variable domain of heavy-chain antibody（VHH）fragments from anti-Enterotoxigenic Escherichia coli（ETEC）flagellar adhesin Fae G protein antibody have been proved to possess sound biological functions.The above VHH fragments were used in this study to be respectively fused into the crystallizable fragment（Fc）region of swine Ig A to form recombinant antibodies of V2A and V3A;the VHH2 and VHH3 fragments were fused into both ends of the Fc region of swine Ig G to form the bifunctional recombinant antibody of V23G;a link was established between the VHH2 and VHH3 to form the bifunctional recombinant antibody of V23.This study was divided into the following three experiments:Experiment 1:The gene sequences expressing the four recombinant antibodies were connected with pPICZαA,the expression vector of Pichia pastoris,to form the recombinant plasmids of pPICZαA-V2A,pPICZαA-V3A,pPICZαA-V23G and pPICZαA-V23.After being linearized,these recombinant plasmids were electroporated into the wild-type of Pichia pastoris X-33 cells to form recombinant yeasts which realized the inducible expression through methanol.The method of ELISA was also employed to screen positive transformants,and the expression products were identified by SDS-PAGE and Western blot.Finally,all recombinant antibodies were purified by affinity chromatography column.The results showed that antibodies of V2A,V3A,V23G,and V23 were successfully expressed in recombinant yeasts.The antibodies of V23G and V23 with high purity were obtained from the supernatant of pPICZαA-V23G-X33 and pPICZαA-V23-X33 recombinant yeasts by using the affinity chromatography column of Protein A and His nickel column respectively,while the antibodies of V2A and V3A were not successfully purified by using affinity chromatography columns of Protein A and Protein G.Experiment 2:Single factor test and orthogonal design test were conducted to determine the influence of four inducible conditions,including the initial concentration of bacteria,the amount of methanol added,the p H of medium and the time of induction,on the expression levels of the four recombinant antibodies.The results showed that optimal inducible conditions of the four recombinant antibodies in Pichia pastoris were different from each other.Within the set interval,the optimal inducible conditions for the antibody of V2A in recombinant yeast were as follows:OD_600nm=20（initial concentration of bacteria）,1.0%（amount of methanol added）,7.0（p H of the medium）,120 h（time of induction）;the optimal inducible conditions for V3A were as follows:OD_600nm=20（initial concentration of bacteria）,1.5%（amount of methanol added）,6.0（p H of the medium）,120 h（time of induction）;the optimal inducible conditions for V23G were as follows:OD_600nm=15（initial concentration of bacteria）,1.5%（amount of methanol added）,7.0（p H of the medium）,120 h（time of induction）;the optimal inducible conditions for V23 were as follows:OD_600nm=20（initial concentration of bacteria）,1.5%（amount of methanol added）,6.0（p H of the medium）,72 h（time of induction）.According to the principle of ELISA,the quantitative methods for these four antibodies were established in this experiment with the protein-gel-extracted V2A and V3A and purified V23G and V23 as standard,respectively.The expression levels of V2A,V3A and V23G in the supernatant of recombinant yeasts were measured by double sandwich ELISA while the expression level of V23 was measured by direct ELISA.It was detected that the expression levels of V2A,V3A,V23G,V23 were 342mg/L,317 mg/L,142 mg/L,and 937 mg/L respectively when optimal inducible conditions were met.Experiment 3:The activities of V2A,V3A,V23G and V23 were explored by whole bacteria antigen-based indirect ELISA and the results showed that all the four recombinant antibodies had binding activity with ETEC.The median effective concentration(EC₅₀)between each one of the four antibodies and ETEC was measured and the levels of EC₅₀ between V2A,V3A,V23G,V23 and ETEC were 9.88μg/m L,8.43μg/m L,9.48μg/m L,and 1.12μg/m L respectively.In summary,the expression vectors for four kinds of swine oral antibodies are successfully constructed in this study and they are successfully expressed in Pichia pastoris.The number of antibody expressions is increased by optimizing the yeast inducible conditions and the four recombinant antibodies all have binding activity with ETEC.