ABSTRACT
Objective SIV30 protein of simian immunodeficiency virus(SIV)was prepared by genetic engineering technique as an antigen diagnostic reagent, to establish an immune comb method for the specific detection of anti SIV IgG in monkey serum. Methods Recombinant expression plasmid of SIV SIV30 gene was constructed by prokaryotic expression vector pGEX-4T-1, and expressed in the competent BL21 cells. The recombinant protein was purified as a diagnostic antigen, and a standardized procedure for the detection of immune comb was established and applied for clinical detection. Results The optimum coating amount of antigen was 0.02 mg/mL. The prepared IC was able to specifically detect the positive serum of SIV. There was no cross reaction between the sera of other viruses. It showed a high specificity of the detection method. Sensitivity analysis showed that the SIV30 protein was able to detect 1:400 times diluted SIV positive sera. The result of stability and repeatability test(the same sample was repeated 3 times) showed that the coefficient of variation(CV)was less than 10%. The serum samples of 10 suspicious monkeys were detected by this method, showing a consistent rate of comb method and ELISA test result of 100%, Kappa =1.000. Conclusions SIV30 protein is expressed in prokaryotic cells. The immune comb is prepared,and is successfullyl applied in clinical examination. It shows that the method has a high sensitivity, strong specificity, good reproducibility and practicability.
ABSTRACT
In order to understand the porcine epidemic diarrhea virus (PEDV) origin and variant characteristics in Liaoning province,diagnosed by PCR,separated by Vero cell,and identified by cell pathological observation,RT-PCR and S gene sequence analysis,1 PEDV strains (LN-2015-1) was successfully isolated from a pig farm of Liaoning province.Analysis of S gene sequence showed that compared withCV777 strain,there were the longest 9 bp insertion,6 bp deletion and 13 bp continuous mutation in addition to point mutation.There also were the longest 3 AA insert,2 AA deletion,and 3 AA or more continuous mutation.The epitope analysis showed that there were 16AA mutations in the 5 epitope regions.Homology analysis show that it had the highest sequence similarity of 99.2％ with HB-HA2015 strain,higher sequence similarity of 98.5％-98.8％ with the domestic and foreign representative strains isolated since 2010,and lower sequence similarity of 93.2％-95.6％ with the traditional strain isolated before 2010;the phylogenetic analysis showed that LN-2015-1 was clustered into the same group with home and abroad variation strain in recent years,and formed a small subgroup with HB-HA2015 at the same time.The evolutionary distance was far from the traditional strains.