| Astrovirus(Astrovirus,AstV)are the single-stranded positive sense RNA virus with no envelope.It mainly infects mammals and poultry.The virus has a high degree of genetic variability and genetics,but also a potential zoonotic disease virus.The main clinical symptom of astrovirus is diarrhea.It is worth mentioning that among all the pathogens that cause viral diarrhea in infants and young children,human astrovirus(HAstV)is considered to be the second largest pathogen,second only to rotavirus.Porcine astrovirus infection rates are high in pigs.It is often mixed with other enteroviruses to cause piglet diarrhea,which is distributed around the world and causes considerable economic losses to the pig industry.At present,there are no relevant reports on the preparation and detection methods of monoclonal antibodies for pig star virus shell protein,so a simple and deliberate test method is needed to diagnose and monitor the disease.This study constructed the expression plasmid pGEX-4T-1-cap-1 by anumbering the highly conservative fragmentof of the coat-shell protein of the specific primer-amplification cloned pig star virus,and transferred to BL21(DE3)recombinant bacteria-induced expression fusion protein,solubility analysis mainly existed in the form of soluble protein,and after using cut gum purification dialysis,SDS-PAGE analytical protein electrophoresis has a high purity,and Western blot detection can bind specifically to GST label antibodies,indicating that fusion proteins have good specificity and antigen.The purified protein was used as the antigen-coated enzyme labeling plate,and the PAstV indirect ELISA detection method was initially established.After the conditions were optimized,the antigen coating amount was finally determined to be 200 ng/well.When OD450≥0.296 determines that the sample is positive,when OD450≤0.267 determines that the sample is negative,and the median value is suspicious.The establishment of indirect ELISA detection method provides an experimental basis for subsequent preparation of monoclonal antibodies.Female mice with purified fusion protein pGEX-4T-1-cap-1 immunity of 6 weeks of age can be used for cell fusion after routine animal immunization procedures.With the purified fusion protein pGEX-4T-1-cap-1 as a package of antigens for indirect ELISA screening positive hybrid tumors,after 5 subclons finally screened 1 stable secretion of Porcine astrovirus pGEX-4T-1-cap-1 protein monoantigen hybrid tumor cells,named F4-4-6.The hybridoma cell line is prepared by ascites to obtain a large amount of antibodies,and the titer of ascites can be measured up to 10~5.By Western blot analysis,the purified antibody can bind to the fusion protein pGEX-4T-1-cap-1,indicating that the antibody has good specificity.With the purified fusion protein pGEX-4T-1-cap-1 as the package antigen,F4-4-6monoantigen as the detection antibody and the serum to be tested competitive solid-phase antigen,established a single anti-competitive ELISA method to detect Porcine astrovirus type Ⅰ antibody.After optimization,the reaction conditions are as follows:the antigen coating amount is 200 ng/well;the blocking condition is 5%BSA at 37℃for 2 h;the reaction condition of the serum to be tested is 1:1 dilution for 40 min;the primary antibody reaction condition is 1:10000 dilution effect for 40 min;anti-antibody reaction condition is 1:5000dilution effect for 60 min;color development time is 15 min.Determine the critical value of yin and yang when the test sample PI≥0.570 determines the sample as positive,when the test sample PI≤0.439 determines the sample as negative,and the intermediate value is the suspicious sample.The specificity and repeatability tests show that the established competitive ELISA detection method has strong specificity and good stability,and can be used for the detection of porcine astrovirus typeⅠserum antibodies. |
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