Study On Biosynthesis Of Polysaccharide Conjugate Vaccine Against Shigella Sonnei

Dysentery is a bacillary dysentery caused by Shigella that is a major symptom of fever,diarrhea,bloody stool and abdominal pain.It is highly contagious,high morbidity and mortality and other serious harm,particularly in children under 5 years of age.Dysentery is mainly caused by Shigella flexneri and Shigella sonnei,and some studies have found that the infection rate of Shigella sonnei is rising in China.In addition,the drug resistance of Shigella is also becoming more and more serious.One of the primary means for controlling infectious diseases is prevention,of which vaccination is considered the most cost-effective measure.Bacterial surface polysaccharides（such as O antigen polysaccharides and capsular polysaccharides）can stimulate the body to produce protective antibodies,however,polysaccharides alone do not produce long-lasting protection and are not effective in children under 2 years of age,If the polysaccharide is attached to a suitable substrate protein to prepare a polysaccharide conjugate vaccine,and it is converted from a T cell-independent antigen to a T cell-dependent antigen,then the deficiency of the individual polysaccharide as a vaccine is compensated.The development and application of polysaccharide conjugate vaccine is obvious to all and is called as one of the most successful vaccines.The existing polysaccharide conjugate vaccines are basically produced by chemical synthesis,there are a variety of defects.Now,in addition to the chemical synthesis of polysaccharide conjugate vaccine,there is a new method—biosynthesis.The purpose of this study is to produce the polysaccharide conjugate vaccine of Shigella sonnei by"one-stop"biosynthesis method,and evaluate its physicochemical,biological characteristics and protectionFirstly,we knocked down the O-antigen ligase（waaL）of S7 strain of sonnei by means of genetic engineering to construct S7ΔwaaL expression strain,and then transferred the plasmid of glycosylation system(pET28a-PglL-CTB^4573C)into the expression strain to produce CTB-OPS sonnei polysaccharide conjugate vaccine in S7ΔwaaL.Secondly,on the basis of this,the substrate protein was replaced with StxB protein whose structure was similar to that of CTB protein to produce StxB-OPS sonnei polysaccharide conjugate vaccine in S7ΔwaaL;At last,we also explored the production of CTB-OPS sonnei polysaccharide conjugate vaccine in E.coli.First,the large plasmid of sonnei was transferred into BL21（DE3）by means of conjugal transfer and named as BSO8,and then transferred the plasmid of glycosylation system into BSO8,the CTB-OPS polysaccharide conjugate vaccine of sonnei was successfully expressed.The results showed that CTB-OPS could be successfully expressed in S7 waaL,and high purity samples could be obtained by purification.Animal immunization experiments showed that CTB-OPS had a protection rate of 72.5%.Although StxB-OPS can express and obtain high-purity samples,but it has been found in animal immunization evaluation that it has little protection against the sonnei and only 10%protection rate,however,It has a 50%protection against Shigella dysenteriae Sd197.In addition,CTB-OPS can also be expressed in BSO8 and obtain high purity samples.This study provides a new idea for the study of the sonnei polysaccharide conjugate vaccine and adds a new members to the Candidate vaccine of sonnei;It also lays the foundation for the further exploration of StxB as a substrate protein for the manufacture of polysaccharide conjugate vaccine;At the same time,it also provides a reference for the synthesis of other pathogenic bacteria polysaccharide conjugate vaccine in E.coli by Biosynthesis.