Two Methods Of Rapid Generating Recombinant Baculoviruses In Escherichia Coli And Functional Characterization Of Orf107 And Orf135 In HaSNPV Genome

The genome of Heliocoverpa armigera single nucleocapsid polyhedrovirus consists of 135 ORFs, 20 of which are unique to the virus. In order to understand the function of the ORFs, two methods of generating recombinant mutants were developed. The methods have become important tools for functional genomic research of HaSNPV. With these two methods, recombinant HaSNPVs with mutations in two unique genes, orf707 and orf735, were constructed and their biological activities were studied. Together with the structure and expression analysis, the putative functions of orf707 and orf735 were characterized.The method of linear homologous recombinant in Escherichia coli was founded on the basis the phage Red system and the bacterial artificial choromosome (BAC). Firstly, the BAC of HaSNPV, HaBacHZ8, was transformed into E. coli strain BW25113 (pKD46) containing the phage Red system. Then a linear fragment with homologue arms of the target gene was transformed into the bacterial, and the homologue recombination occurred with the help of the recombinase. As the recombination process takes place in the prokaryotic system, it greatly reduces the time and expense of the traditional method of co-transfection/plaque purification. This method was further improved by constructing linear fragment with PCR products, which simplified the process significantly.Another method is to make a random insertion library. A random mutant library of HaBacHZ8 containing 1300 clones was constructed with transpon TnMaxl3. Target screening was established by primary selection of three rounds of PCR and further identification with PCR and sequencing. The successful screenings of many target mutants have demonstrated that the library was successful.The truncated orf707 was cloned and expressed as a 20kDa protein in E coli. The antiserum was derived by immunizing rabbits with this protein. Western blot analysis using the antiserum revealed a protein larger than 170kDa in the HaSNPV infected cells. The studies of orf707 deletion mutant HaBacHSl and insertion mutant HaBacHS2 indicated that or/707 is an un-necessary gene for HaSNPV infection.The orf135 was expressed as a 28kDa protein by prokaryotic expression vector, and the antiserum was produced in rabbits. Western blot analysis revealed a specific protein band with a size of 26kDa from the HaSNPV-infected HzAMl cells at 24h to 72h post infection. The study of orf135 deletion mutant indicated that orf135 plays an important role in the life cycle of HaSNPV.