Expression Of Surface Specific Antigen Of Echovirus And Establishment Of Rapid Detection Technique

Enteric Cytopathogenichuman Orphan Virus(ECHO),simply called echovirus(E),is single strand RNA virus that belong to the genus Enterovirus of the Picornaviridae family which are divided into 34 species.Echoviruses are widely spread in the crowd all over the world through respiratory tract and digestive tract,results in numbers of human diseases,among which the aseptic encephalitis is the most common one.Of all these serotypes of echovirus,a lot of studies focused on the echovirus type 1(E1),E6 and E9 due to their strong infectivity and pathogenicity.However,considering their increasing upward trend and severity,more attention shall focus on this issue,which means we need more effective and faster way to test their infection within a short period.Currently,echovirus detection methods include virus isolation culture method,enzyme-linked immunosorbent assay(ELISA)and reverse transcription PCR method.However,there are several obvious disadvantages of these methods like high costs,requirement of special equipment,time-consuming,complex treatment of samples and etc.Since these facts have badly limited the practical application range,we need some new technology of testing,to simplify testing processes,save time as well as testing expenses.Gold Immunochromatographic Assay(GICA)is a promising method for its advantages like quick testing;easily applied to field,needless of special equipment,simplified processes,and easily observed&understood results.The key issue of developing this method is to find an ideal specific antigen of echovirus.VP1 proteinof echovirus is a very important kind of surface protein of echovirus.Generally,almost every subtype of echo virus has surface protein VP1;it is closely related to antigenic determinant,which is the key factor to tell different serotypes and determine virus neutralization.Its conservative property is good for vaccine development and makes it possible of being detection reagent.Currently all gene sequence testing work for VP1 of echovirus has been done,which is good for following diagnostic reagent and vaccine developing work combined with gene engineering technology.This study takes 3 surface proteins VP1,i.e.E1,E6 and E9 as research subjects.Firstly,we analyzed the epitope of VP1 by computer software and then screened epitope-rich region.The gene sequence was optimized according to preferred prokaryotic gene codon,chemically synthesized and then cloned into pGEX-4T-2 and pET-28a(+)plasmids,getting six recombinant expression vectors.The six recombinant expression vectors were then transformed into E.coli BL21(DE3)respectively and engineered bacteria with high expression of target protein were screened.The six kinds of VP1 fragments were purified by affinity chromatography as E1-VP1-GST,E6-VP1-GST,E9-VP1-GST,E1-VP 1-His,E6-VP1-His,E9-VP1-His.Secondly,three VP1 fragments with His-tag were used to immunize rabbits,to produce polyclonal antibodies and then to detect titer of polyclonal antibody.Then three purified VP1 fragments with GST-tag conjugated to colloidal gold were added onto glass fiber membrane.Rabbit anti-human polyclonal antibodies(IgG&IgM)and the produced polyclonal antibodies were respectively added to the test line and the control line of nitrocellulose membranes.Combined with double-antibody sandwich method,four different colloidal gold test strips,including one mixed using strips and three single using strips were assembled for detecting.Afterwards,using these strips to test different dilution ratio of a specific concentration of positive serum of echovirus infected patients,getting maximum dilution ratio is 1:64 to prove it has good sensitivity.Meanwhile,the strips also give a result of negativity to show its specificity after testing with positive serum of other similarly virus infected patient.Compared with traditional ELISA technique,these two methods has no statistical difference in final results,however,the new one gives quicker result in 10 minutes.These new strips fulfill the testing requirements of sensitivity,specificity as well as clinical detection,and the characteristic of the large number detection is more suitable for the rapid detection of the needs of the field,which leading more important realistic meaning and pretty high commercial value.