Amplification And Cloning Of The Gene Of Encoding Fusion Protein Of Newcastle Disease Virus And Expression Of Its Fragment In E.Coli

The gene encoding fusion protein of NDV was amplified, cloned and expressed with molecular biological techniques. Fusion protein is the main protective antigen of NDV and the primary structure of fusion protein determines the virulence of the virus. Hence, the molecular basis of the gene encoding fusion protein of NDV is of great importance for construction of NDV genetic engineering vaccine and its actual utilization.RT-PCR method used for amplifying F gene of NDV and sequencing of the complete gene and comparison with published NDV F gene of F48E9 strain. The result of deduced amino acid sequence of fusion protein indicated that the NL isolate was a virulent virus, which was homologous to the publishing NDV F48E9 strain by 87.81 percent in nueclotide sequence of F gene and by 92. 01 percent in amino acid sequence of fusion protein. The 112th amino acid in the cleavage site of NL isolate is K, while F48E9 strain is R. It indicates that the NL isolate is a virluent virus which differs from F48E9 strain.On the basis of recombinant PCR principle, two fragments of F gene from NL isolate were ligated together using PCR technique and inserted into T-vector, which was further transformed into E. coli TM109 strain. It was named PFL plasmid. Identification by means of restriction enzymes and partial sequencing which demonstrated that the cloned gene of complete DNA sequence contains starting and ending codons of translation and the leader sequence for beginning of transcription and it is possible to be used for expression in prokaryote and eukaryote expression systems.In order to study on NDV genetic engineering vaccine, we designed a pair of primers with enzyme sites On the basis of sequence of F gene of NL isolate and amplified PFL plasmid using PCR. We got a 630 base pair fraction which identifying with the result of designing. Then it was inserted into the Xbal /Xhol sites of pThioHisB vector DNA. The resulting recombinant pThioHisB/F was determined by means of restriction enzymes and PCR. It indicated that the cloning was correct. It was transferred into E.coli toplO cell inducing with IPTG. A specific expression product of NDV F protein with a molecular weight of 35 KD was detected by SDS-PAGE and western-blot analysis. The expressing quantity'of fusion protein is about 20 percent of the cell total protein.