| Gonorrhea caused by Neisseria gonorrhoeae is one of the sexually transmitted diseases with highest incidence in the world.N.gonorrhoeae infections can lead to infertility,but also promote the spread of human immunodeficiency virus(HIV).The range of drug resistance of N.gonorrhoeae is constantly expanding with the update of types of antibiotics,which brings great difficulty to the treatment of gonorrhea.So we need a comprehensive understanding of the biological characteristics of N.gonorrhoeae,in order to facilitate the fundamental control of gonorrhea.Clustered regularly interspaced short palindromic repeats(CRISPR)consisting of the CRISPR cluster,leader sequence(Leader)and a series of CRISPR related protein(Cas)genes are acquired immune system that presents in 40%of the bacteria and 90%of the archaea.And it plays important roles in preventing phage and foreign gene invasion.Our previous studies revealed that 3 CRISPR clusters and 2 Cas genes existed in the genome of N.gonorrhoeae WHO-A strain.The 2 Cas genes belonged to the CT1133 family and the TM1801 family.In the present study,the expressions and functions of gonococcal Cas genes were explored.The studies were divided into 4 parts as following:Part one:Typing of gonococcal Cas genes and construction of prokaryotic expression vectorsAt present,at least 45 Cas gene families were reported.In order to analyze the genotype of Cas gene of gonococcal WHO-A strain,the CT and TM Cas genes in the strain were analyzed by BLAST to reveal the homology with published genes.To reveal the structure and function of Cas proteins in gonococcal CRISPR system,the specific location of Cas genes were determined according to the results of genome sequence of WHO-A strain.The rimers were designed to clone the CT or TM Cas genes.Digestion sites of Pst I and Kpn I or BamH I and Xho I were added to the upstream and downstream primers to insert CT or TM genes into pCold-TF and pGEX-6p-1,respectively.Cas genes were amplified by PCR with the template of the genome DNA of WHO-A strain.The amplified products and the vectors were digested with the same double restriction enzymes and ligated by T4 ligase to get the prokaryotic expression vectors.Part two:Preparation of polyclonal antibodies by using recombinant Cas proteinsThe prokaryotic expression vectors pCold-CT(TM)or pGEX-CT(TM)were transformed into BL21 E.coli,and the recombinant strain was induced by IPTG.Following detection the molecular weight by SDS-PAGE,supernatants of induced products were purified by nickel affinity chromatography column or glutathione resin column.The purified products of CT-His,TM-His,CT-GST,and TM-GST were analyzed by SDS-PAGE and BCA kit to get purity concentration of recombinant proteins.BALB/c mice were immunized with purified fusion protein of CT-His or TM-His.Immune serum was collected 4 weeks after the latest immunization.The titer of specific antibody in immune serum was detected by ELASA using CT-GST or TM-GST fusion protein as antigen.The results showed that CT and TM recombinant proteins were obtained with the expected molecular weight.The titers of antibodies of CT and TM in the immune serum were 1:25600 and 1:12800,respectively.The specific antibodies provided materials to analyze the expressions and functions of Cas proteins in gonococcal WHO-A strain.Part three:Expression analysis of gonococcal Cas genesTo reveal the transcription and expression of Cas gene in WHO-A strain of N.gonorrhoeae,Cas-mRNA and Cas protein were detected by RT-PCR,ELISA and immunofluorescence,respectively.?.The total RNA extraction from the strain was reverse transcripted to the cDNA which was amplified by PCR.The PCR product was analyzed by 1%agarose gel electrophoresis.?.Cas proteins in gonococcal lysate were determinated by chessboard titration with the prepared CT or TM immune serum as primary antibody.?.The WHO-A strain cells were fixed on the slide with 4%paraformaldehyde.The CT or TM immune serum was used as primary antibody while FITC-labeled goat anti-mouse IgG was used as secondary antibody for immunofluorescence assay with fluorescence microscopy.The results showed that the DNA bands with the size of CT or TM gene could be amplified by RT-PCR from the total RNA of WHO-A strain.When total proteins in bacterial lysate of WHO-A strain was diluted to 200 ?g/ml,a specific signal of the expression of Cas protein was detected with 1:12800 CT polyclonal antibodies or 1:800 TM polyclonal antibodies.Bacterial cells of WHO-A strain could be detected the specific fluorescence with CT or TM antibodies.These results suggest that the Cas genes were effectively transcribed and expressed in the WHO-A strain of N.gonorrhoeae.The CRISPR system of N.gonorrhoeae was a complete and active system,so the structure and function of Cas proteins should be further studied.Part Four:A preliminary study on functions of Cas genes of gonococcal WHO-A strainThe pCas9-KRn,a simplified CRISPR system targeting the kanamycin resistance gene,has been constructed in the laboratory and could be used as a tool for the study of the activity of Cas proteins.To investigate whether the CT or TM protein exerts nuclease activity,the Cas9 gene in pCas9-KRn was replaced by the CT or TM gene.PCR amplification was used to get a vector frame without Cas9 gene from pCas9-KRn.The PCR product digested with Pst I and Kpn I endonucleases was ligated to CT or TM gene that was digested with the same endonucleases to obtain the recombinant plasmid pCT(TM)-KRn.Following transformation the recombinant plasmids to BL21(DE3)E.coli,the expression of WHO-Cas genes was detected by R.T-PCR and ELISA.The recombinant vector of pCT(TM)-KRn was transformed into pET-30a containing DH5? E.coli.The recombinant strain was passaged with LB medium.The results of viable bacteria on kanamycin or chloramphenicol LB plates showed that there was no significant difference in CFU between the 2 antibiotics plate,suggesting that no nuclease activity of WHO-Cas protein was found.The role of gonococcal Cas protein was different from the nuclease activity of Cas9. |
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