| Shigella are intestinal bacteria that can cause diarrhea in adults and children after infected. Previously it was believed that Shigella can not make livestock disease,in fact,a large number of domestic and foreign related news show that Shigella can infect a variety of animals,such as pigs,foxes,monkeys,cows,chickens,ducks,etc.,it can cause diarrhea, abdominal pain,even lead to death.Recently it was discovered chicken shigellosis is an acute infectious disease that can lead to different ages and different varieties of chicken diarrhea.This disease is widespread and harmfull to the healthy development of the poultry industry and has brought huge economic losses for poultry industry. Shigella may has a very wide range of cross-infection between human and animals,that can seriously endanger human public health and veterinary public health.Shigella invaded into intestinal epithelial cells is the key of shigella pathogenic.Numerous studies show that Ipa C can induce the cytoskeletal rearrangements that allow Shigella invade into epithelial cells,Ipa C is secreted by the type III secretion system(TTSS). It is proved that only recombinant purified protein Ipa C could be able to recover the invasive ability of Shigella. At the molecular level, Ipa C is shown to possess a distinct functional organization. Its N-terminal 20 amino acids provide a signal for type III secretion.Ipa C’s central hydrophobic region has two putative transmembrane helices that are responsible for its penetration of phospholipid membranes and contribute to overall protein stability in the absence of phospholipids.The Ipa C C-terminus possesses a putative coiled-coil domain that may be involved in oligomerization.The C-terminus of Ipa C related to trigger actin.Chicken and Human Shigella have the same Ipa C protein molecules, and molecular phylogenetic analysis indicat that both of them may have a common evolutionary origin.Therefore,it is important and have great significance to use the yeast expression system in order to maintain the native conformation of Chicken Shigella Ipa C.In this study, take original sequence gene Ipa C and codon optimized synthetic gene Ipa C * as research objects,constructded eukaryotic expression vector p GAPZαA-Ipa C and p GAPZαA-Ipa C * and transformed them into yeast host strain X-33 by electric shock.With western blot analysis,it was proved that Chicken Shigella Ipa C * gene was successfully expressed in Pichia pastoris.The results are as follows:1. According to p GAPZαA multiple cloning site and Chicken Shigella Ipa C gene sequence to design a pair of primers. Take the recombinant vector p ET32a-Ipa C saved in our laboratory as a template, amplified Chicken Shigella Ipa C gene by PCR.Both the Ipa C and eukaryotic expression vector p GAPZαA digested,and transformed into E. coli TOP10.It was detected by bacteria PCR and restriction enzyme digestion, sequencing of plasmid.All of them shown that it was successfully constructed the eukaryotic expression vector p GAPZαA-Ipa C.2.Eukaryotic expression vector PGAPZαA-Ipa C after single enzyme linearized, electroporation transformed into yeast host strain X-33,then picked and expanded the high resistance positive clones from(Zeocin concentration of 2000 ug / m L) YPD solid medium culture.Identify them with yeast genomic PCR amplification and sequencing, picked the correct recombinant yeast p GAPZαA-Ipa C / X-33 into YPD liquid medium, Regular sampling at different time points, with SDS-PAGE and Western blot analysis showed that Shigella Ipa C chicken genes were not expressed in the X-33 intracellular and extracellular.3.Under the condition of without changing the Ipa C protein amino acid sequence,according to Pichia codon to optimized synthetic Chicken Shigella Ipa C * gene. Cloned Ipa C* into the yeast expression vector p GAPZαA and transformed into E. coli TOP10, and choose the right recombinant plasmid p GAPZαA-Ipa C * transformed into yeast host strain X-33, and detected by the high resistance screening, PCR amplification and sequencing. Picked the correct clones into YPD liquid medium, regular sampling at different time points and detected with SDS-PAGE electrophoresis. The results showed that culture supernatant after ultrafiltration concentration had no obvious target stripe,but the cell precipitate had target stripe in the 70 k Da. The expressing protein product detected respectively with Ipa C immune antibody serum, Chicken Shigella bacteria inactivated vaccine immune antibody serum and His tag antibody use the Western blot analysis. All of them had specific band in the 70 k Da.It indicating that the optimized synthetic Ipa C * gene was successfully expressed in yeast intracellular. It verifying that the expression level of yeast expression system relate to exogenous gene. Recombinant protein expression in this study is about of 70 k Da, the maximum amount expression at 108 h,it was Intracellular expression of yeast. |
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