Cloning And Sequence Analysis Of The Partial Genome Of GPV And MPV And Expression Of VP2-VP3 Gene

Cloning and Sequence Analysis of the partial genome of GPV and MPV and Expression of VP2-VP3 GenePh D Candidate: Li Xuemei Tutor: Fei Enge Associate Tutor: Jin NingyiAbstract Goose Parvovirus (GPV) and Muscovy Duck Parvovirus (MPV) infection is two kinds of highly contagious and deadly diseases for goslings and Muscovy ducklings. Both of GPV and MPV have been classified as the members of the autonomous parvoviuses. Their genomes show high similarity, and share 81.9% nucleotide sequence identity. The biological properties of GPV and MPV isolates from different areas in China were studied. They were identified by virus culture characteristics, virus purification (CsCl gradient centrifugation), electron microscope (EM) technique and Agar gel Precipitation test.We designed and synthesized two pairs of primers according to nucleotide suquences of GPV B strain and MPV FM strain, and mutated the VP2 ACG codon to ATG one (bold T in the upper primer). The complete VP2-VP3 (the major structural protein) genes of four domestic isolation stains (GPV CC, GPV FS, MPV YZ and MPV FS) were amplified by PCR, and obtained about 1.8kb fragment. Four recombinant plasmids (pGVPCl, pGVPFl, pMVPYl, and pMVPFl) were constructed by cloning PCR products into pCR3.1 T vector respectively. The sequence analysis showed that nucleotide sequences of VP2-VP3 genes of three strains consist of 1764nt, encoding 588 amino acids. Surprisingly, the VP2-VP3 gene of GPV FS strain deleted 1 base. Comparing two isolates (GPV CC and GPV FS) with GPV A, we found that their nucleotide sequences homologies were 98.9% and 93.0%, amino acids sequence homologies were 98.6% and 96.3%. These results indicated that GPV CC strain is closely related to GPV A strain. In addition, we also compared these strains nucleotide sequences homologies and amino acids sequence homologies. The fact was found that there is a higher relevance among these strains (MPV YZ and MPV FS show 99.3%, 98.4% nucleotide sequences similarity and 98.3%, 96.5% amino acids sequence similarity to MPV FM). But GPV strain show 78.3%?0.6% nucleotide sequence and 84.8%~87.6% amino acids similarity to MPV.Another two pairs of primers used to amplify the complete NS (nonstructural protein) gene of both of GPV and MPV, were designed and synthesized. The NS genes of four domestic isolated strains (GPV CC, GPV FS, MPV YZ and MPV FS) were obtained by PCR and sequenced after being directly inserted into the pCR3.1 T vector. The sequence analysis demonstrated that the nucleotide sequences of NS genes of all of strains were1884bp, encoding 628 amino acids. Comparison of two isolates (GPV CC and GPV FS) with GPV B strain, the NS genes percentage identities at the necleotide sequence level and the amino acid level were 98.3%, 92.7% and 99.4% respectively, and were 92.5% and 94.6% between two isolates. We also compared the NS genes of three MPV strains each other, the result showed that the degree of identity at the nucleotide sequence level and the amino acid level were 99.3%, 98.3%, 98.5% and 98.4%, 96.5%, 97.3%(MPV YZ to MPV, MPV FS to MPV FM and MPV YZ to MPV FS). These results suggested that the homology (79.6%~83.0%) of nucleotide sequences and amino acids sequence of the NS gene of MPV strains were higher than that (86.9%~-90.7%) of VP2-VP3 gene.The recombinant plasmid pGVPCl was digested with BamH 1, and the resulting fragment was inserted into the BamH I site of the pET-28a under the downstream sequences.Another prokaryotic expression plasmids pEMVP was constructed successfully by inserting VP2-VP3 gene of MPV ZY strain into the BamH I and Xho I site of pET-28a. E. Coli component host BL21 was transformed with two expression plasmids. Both of the recombinant bacteria could express the VP2-VP3 gene after induced by IPTG, and the recombinant proteins were analyzed on 12% SDS-PAGE and stained with Comass brilliant blue or detected by immunblotting analysis with anti-GPV (or anti-MPV) serum which were prepared in rabbits immumized with purified GPV CC (or MPV YZ).Two Eukaryoti...