| White spot syndrome virus(WSSV) can cause white spot syndrome(WSS), which has widely host and presently causes the serious losses to shrimp farmers worldwide. At present, there are only 3 full WSSV genomes sequences at GenBank(AF369029 from Thailand, AF332093 from China, and AF440570 from Taiwan). The homology of the 3 sequences is reached up to 99.32%. However, there are also a few sequence variations, one of which is variable number of tandem repeats(VNTR). In order to investigate the diversity of VNTR in ORF94, the virus was isolated from Jiangsu and Zhejiang Province then their genes were amplifued, their sequences were aligned, and VNTRs sequences were analyzed. Furthermore, our objective was to assess the connection between the VNTRs and the virulence of 2 different WSSV virus which have the most disparity by comparing mortality patterns of Procambarus clarkii of the same size class following intramuscular inoculation with same infections dose of each isolate. In addition, envelop of virus plays an important role in their activities. However, there are limited researches on the interactions between envelop proteins of WSSV so far, due to the lack of effective cell line. VP28 is one of the major envelop proteins of WSSV, and it participate in their attachment to and penetration into shrimp cells. In this study, the recombinant protein VP28, VP31 and VP36A were expressed and purified, and the virus was also purified and fracted. To prove the interactions between VP28 and VP31,VP36A, here we carried out far-western blotting assays and His pull-down experiments.Based on the genome sequence of WSSV from Thailand, primers were designed to amplify whole ORF94 gene of five virus from Jiangsu and Zhejiang Province with PCR. The PCR product was cloned into pMD 18-T vector routinely, and the positive recombinants were identified by ampicillin screening and endonuclease digestion. The correct positive ecombinants were then sequenced and analyzed. The homology of nucleotide between the five virus and the other virus at GenBank is up tp 95%. The number of VNTR is from 2 to 14, and the polymorphic site was significant different. Then two WSSV isolates were used in this study. All 2 isolates were collected from naturally infected shrimp, one from Ganyu and the other from Dafeng. The lethal median dose of these stocks of gill suspension, as determined by intramuscular inoculation of Procambarus clarkii, was 10-5.6 and 10-5.5 for Ganyu and Dafeng, respectively. Then Shrimp were inoculated intramuscularly with 2mL of inoculum containing 1 LD50 of each isolate in the junction between the third and fourth abdominal segments. Following inoculation with the 2 different isolates, shrimp were monitored and recorded for signs of WSS, including anorexia and lethargy, every 12 h until the end of each bioassay. Dead shrimp were collected 2 times per day, and a part of them were stored in -40℃. At the same time, sequences of ORF94 of dead shrimp were analysed and cells and virus were observed with electron microscope. There was no difference between VNTR, and the diversity of the polymorphic site is not significant. Therefore, it was presumed that there was little connection between virulence and VNTR of ORF94.Purification of virus was performed as described previously. Viral envelope was separated from the nucleocapsids and extracted by Nonidet P-40, Triton X-114 and acetone. Based on the genome sequence of WSSV Ganyu, primers were designed to amplify VP28, VP31 and VP36A by PCR. The products were first cloned into pMD 18-T vector, then inserted to plasmid pET-32a for making recombinant plasmid and then transformed to host strain BL21. Both of recombinants were well expressed in the form of inclusion body with 1.0 mmol/L IPTG at 37℃. According to the results of SDS-PAGE, the recombinat proteins were 28kDa,57kDa and 62kDa, as expected. Far-Western and Pull-Down assay experiments showed that there were no interactions between VP28 and VP31 or VP36A. |
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