Rescue And Biological Characteristics Study Of A Holin Gene Deletion Recombinant T7 Phage

Because of its simple structure and convenient for operation,T7 bacteriophage is widely used in various fields of veterinary research for rapidly and conveniently screening of antigens and antibodies as a tool of the phage display technology.Based on its lysis mechanism,this study construct a recombinant T7 phage?T7-?Holin?which lacks the lytic regular gene?Holin?to explore the correspondence between the T7 phage lytic cycle and the progeny virus particle yield.Through the establishment of a evaluation method that based on the fluorescence quantitative PCR,the process parameters of the culture process was optimized,a high-efficiency culture method for the holin-deficient strain T7 phage was finally determined,laying the foundation for large-scale,low-cost production of T7 bacteriophage.First research was splicing of T7 phage genome with deletion of holin gene by PCR and construction of a compensatory host expressing the holin protein to rescue the recombinant T7-Holin phage.The Holin gene was amplified by PCR with which Xba I and RBS sequences added at the 5'end and BamH I site added at the 3'end respectively,then insert it into vector pET-28 to construct the recombinant plasmid pET-Holin.Transferred the recombinant plasmid into E.coli BL-21?DE3?by chemical transformation to construct the compensatory BL21-Holin host strain.The Holin protein was expressed by IPTG induction and identified by SDS-PAGE.Select the Sfi I site in upstream and the non-coding region in downstream of the holin gene as the segmentation sites by analyzation of the whole genome of T7 bacteriophage.The left genome of Sfi I site was cut by restriction enzyme and recovered by gel extraction,and SOE-PCR method was used to amplify the fragment from Sfi I sit to non-coding region with Holin gene deletion,while the right genome of non-coding region was amplified by conventional PCR.The junction fragment of these 3 PCR productions was packaged by packaging protein and rescued in the compensatory host.The results showed that the compensatory host could express the Holin protein correctly and the recombinant phage T7-?Holin was successfully rescued.The sequencing result showed that the Holin gene was correctly knocked out and good genetic stability was showed as the deletion of Holin gene could stably transmit for at least 15 generations.Due to the disadvantages of low efficiency,low flux and inaccurate results in evaluating the total number of phage particles by the traditional plaque counting method,in the second research,a better evaluation method which based on the qRT-PCR was established in order to better study the biological characteristic of T7-?Holin.Firstly purified T7 phage particles were obtained by gradient density centrifugation and used as template in qRT-PCR after serial dilution to established standard curve and a formula which between the CP value and the concentration of purified T7-?Holin samples.Use ultra-micro spectrophotometer to measure the absorbance of the samples that has been serial diluted,number of phage particles in the samples could be calculated by a emprical formula that between the sample concentration and the number of phage particles.Convert the sample concentration into CP value through the first formula,then the number of phage particles in the sample can be efficiently,quickly and accurately calculated through the formula between the CP value and the number of particles.The plot showed that the established CP value-particle number equation has a good linear relationship,which means the evaluation established in this experiment can be used to assess the number of phage particles in the test sample by qRT-PCR quickly,accurately and with high throughput.In the third experiment,the differences in growth rate,lysis time and progeny yield of T7-?Holin and T7Select415-1b were compared by changing the MOI and the condition of IPTG,then plotting the growth curve by time-shared sampling.It was found that the growth rate of T7-?Holin was slower than that of T7Select415-1b and the lysis time was delayed by about half an hour and the number of progeny particles was about 5-fold higher than that of T7Select415-1b when MOI>1.The cleavage time of T7-?Holin was delayed more and more with the decrease of MOI when MOI<1.T7Select415-1b was completely lysed after 4h but T7-?Holin has no remarkable lysed feature after more than 5h when MOI was 10^-5,while the progeny yield of T7Select415-1b and T7-?Holin was almost the same under low MOI.The results also showed that changing the concentration and adding time of IPTG had no effect on the growth and lysis time of T7-?Holin.RNase and DNase treated samples and non-treated samples were evaluated by qRT-PCR evaluation respectively.The results showed that the particles in T7Select415-1b samples before treatment was about two titers higher than that after treatment,while the difference that between T7-?Holin samples with and without treatment was only one titer.The particles of samples acquired after culturing for a long period of time tend to be uniform,showing the free nucleic acid released during the cleavage of T7 phage could have some effect on the evaluation method that based on the qRT-PCR.