| Porcine Rotavirus(PRV) belongs to the family reoviridae. It is one of the major pathogens of diarrhea in piglets and causes a major health worldwide in graziery. Infection of PRV is also prevalent in china, in some area the infectious ratio of piglets is 72%. Accurate diagnosis and effective vaccination are main means for the prevention of PRV diarrhea.In this study, porcine rotavirus was successfully isolated from diarrhea! piglet fecal samples from Jilin province. MA 104 cells were used for the replication and isolation of the virus. After four passages, the isolate could produce obvious cytopahic effects (CPE). Typical rotavirus particles were seen with direct electron microscope. Polyacrylamide gel electrophoresis (PAGE) of its dsRNA genome showed its RNA electropherotype was 4:2:3:2 which accords with the electropherotype of guoup A rotavirus. The isolate was designated JL94.The plague characteristic of JL94 was studied with double layer method. It could form distinguishable plagues in MA104 cells. After incubated for 5 days, the majority of plagues was about 1mm in diameter. The shapes of plagues were difform and some of them were round. The plagues were no color under low power microscope and the area of the plagues consisted of obtrite dead cell lumps.The hemagglutinating characteristic of JL94 was studied tentatively with human and 9 kinds of animal red blood cell(RBC). It revealed that cell culture of JL94 hadn't agglutination to human group O, A, B and chicken, goose, simian, mice, guinea pig, goat, dog RBC. Its agglutinatioin to porcine and rabbit RBC was labile and needed to study continually.According to the reported complete VP6 nucleotide sequences of group A PRV in GenBank, a pair of primers was designed to amplify VP6 gene of JL94 with Oligo 4.1 software. The segments of complete gene 6 of JL94 were obtained from RT-PCR using dsRNA extracted from the virus as template. The product of PCR named VP6 is approximate 1.3kb in length. The VP6 gene was cloned into pMD18-T vector and sequenced. The results of sequencing showed that JL94 isolate complete gene 6 was 1356bp and had a complete open reading frame which encoded 397 amino acides. The sequence was analyzed homologously in comparison with the VP6 nucleotide sequences of two reference porcine rotavirus OSU(subgroup I ) and Gottfried (subgroup II). Nucleotide sequence comparison indicated that JL94 shared 86.85% and 82.41% identities with OSU and Gottfried respectively. The deduced animo acid sequences were 97.84% and 93.20% correspondingly. The result affirmed that JL94 isolate is more similar to OSU than Gottfired in Gene type.The VP6 gene was subcloned from recombinant plasmid pMD18-T-VP6 into expression plasmid pET-30a. The recombinant plasmid pET-30a-VP6 was transformed into E. coli BL21(DE3) and induced with IPTG. Not only a fusion protein about 45ku as we expected was found but also several smaller polypeptides were observed. The amount of the goal protein was evaluated by densitometric scanning. It indicated that the 45ku product of the VP6 gene was 26.5% of total bacterial protein of BL21. The expressed products were purified by metal(Ni2+) chelation affinity chromatography and tested by western-blot. The results showed that His-tagged VP6 fusion proteins have a positive reaction with anti-JL94 serum.This study provides the basis evidence and material basis for PRV identification molecular epdiemiology investigation , research of diagnostic reagent and genetic engineering vaccine. |
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