Cloning And Construction Of The Prokaryotic Expressing Vector Of Partial Gene Of Classical Swine Fever Virus

Swine Fever (SF), caused by Classical Swine Fever Virus (CSFV), is an extensive popular and high contagious disease. Measures to control and eradicate SF have been taken in many countries, but Swine Fever is still one of the first contagious diseases, which threaten swine industry nowadays, due to globalization of swine trading and intensivism of swine breeding.With the development and broad application of Chinese lapinized vaccine of CSFV, the outbreak of Hog cholera has become random and regional which was extremely prevalent in the past, and the course of disease has become chronic, non-typical Hog cholera emerged at the same time. The objects infected are mainly pregnant sow and piglet, accompanying with many other characteristics such as persistent infection,virus syndrome with pregnant sow infected, sow progenitive obstacle, and fremitus in piglet innated.Immuned swinery infected with CSFV appeared.In order to build an accurate and quick diagnostic technique, to analyze if there is the antigenic gene of vaccine mutated in our country nowadays, and to set up a convenient method to make clear that if the antibody in the infected swine is caused by vaccination, in this research a RT-PCR technique was primarily established to detect CSFV, obtain the gene of epitope on E2 protein, and successfully construct a prokaryotic expression recombinant plasmid to express protein of E2.At first, according to the CSFV genomic sequence accessed in GeneBank and the research by eldership, a pair of primers were designed as following: PC (5'-CGGAGGGAC TAGCCRTAGTGG-3'), PD (5'-GTACACCGGTTCCTCCACTCCCAT-3'). Total RNA were extracted from the infected spleen of rabbit, PK-15 cell, spermary cell of calf, primary kidney cell of porket, primary kidney cell of rabbit, and the supernatant of the above-mentioned cell cultures respectively, and then the gene fragment was amplified by RT-PCR. The experiment proved that RNA from infected kidney of rabbit, centrifugal supernatant of infected PK-15 cell and spermary cell of calf was served as amplified templet, and the amplified genome (P_CD) is about 326bp, consistent with the one reported in the Genbank. Identified by Restriction Endonuclease Reaction, expectant restriction endonuclease sits can be found. Truncated fragment was ligated to Vector of PMD 18-T, and sequenced with blue and white screen, bacterial culture PCR and plasmid PCR.The result showed that ratio of sequence similarity with lapinized Chinese strain of AF531433, AY805221, AY382481, AF091507 and Z46258 are 99.4%, 99.4%, 99.1%, 95.8%, 97.8% respectively, virulent strain Brescia of M31768 and AF091661 are 96.3%, 98.5%, respectively, virulent strain Alfort/187 of X87938 are 98.2%. All these indicated that expectant fragment was successfully amplified and RT-PCR method to diagnose CSFV was established.Secondly, based on the reported genomic sequence of CSFV in the GeneBank and multi-clone sites of expressive plasmid pGEX-4T-1,another couple of primers(A:5'-CGG AATTCTTAACTAGGGTCTGGAATAGCG-3', B:5'-GCGTCGACCCACTGGCTCGCCTTTCACA-3') were designed, which were used to prokaryotically express antigenic region of E2 protein. Restriction Endonuclease sites and protective base pairs were appended to 5' terminal end of primers in order to express easily. A fragment about 703bp was obtained from supernatants culture infected PK-15 cell by RT-PCR, which is consistent with what were expected. Then the fragment was ligated into PMD 18-T vector; to sequence it after identification with bacterial culture PCR and plasmid PCR. The result indicated that the similarity in sequence with AF531433,AY805221,AY382481,AF091507,Z46258,M31768,AF091661,X87939 are 99.6%,99.4%,99.1%,96.0%,98.9%, 89.9%,92.6%, 95.6% respectively, different base pair distributing randomly in the whole sequence, and there are no base gap or base insert, based on genomic sequence ,we can conclude the similarity of amino acid is 100%,99.6%,99.6%,95.3%,99.6%,90.2%,92.3 %, 96.2% respectively.At last, PE2 gene has been cloned into prokaryotic expression vector pGEX-4T-1 to construct the recombinant vector by recombinant DNA technique and the recombinant plasmid has been transformed into E.coli BL21 in order to obtain PE2 protein acting as immune antigen to prepare monoclonal antibody. Recombinant plasmid pGEX-4T-1-PE2 is obtained by bacterial culture PCR/plasmid PCR/double restriction endonuclease.In a word, in this research, a RT-PCR method has been established to diagnose CSFV and clone gene fragment of antigenic region from E2 protein of lapinized Chinese strain of CSFV, and to construct recombinant plasmid for prokaryotic expression successfully. All these will offer some experimental foundations for further researching CSF and CSFV.