| [Background]Urogenital tract infection caused by Chlamydia trachomatis(Ct)is a kind of sexually transmitted diseases(STDS)with the highest incidence rate at home and abroad,numerous complications and difficult to recover from repeated course.According to the statistics on reproductive tract chlamydia trachomatis infection of Centers for Disease Control and Prevention(CDC),the rate of Ct infection among females(692.7 per 100,000)was about two times the rate among males(380.6 per100,000)in 2018.Since most Ct infection patients are asymptomatic,the initial damage caused by Ct infections is usually overlooked,especially in women with higher infection rates,which can lead to serious complications such as pelvic inflammatory disease and ectopic pregnancy.In recent years,the effective inhibition rate of antibiotics on Ct infection has shown a decreasing trend.The effective vaccine against Ct infection hasn’t been reported yet,although animal models and human studies have shown that it is feasible.Phage therapy has been used clinically in the treatment of chronic,persistent,or antibiotic-resistant bacterial infections for many years,but phage specific to Ct has not been discovered yet.Previous studies of our laboratory found that Guinea Pig Inclusion Conjunctivitis Chlamydia(GPIC)phage PhiCPG1 capsid protein VP1 has a significant inhibitory effect on Ct infection both in vivo and in vitro.We predicted through the Inter Pro website that the 18-476 amino acid sites were the functional domain of VP1 protein and constructed the functional domain truncated protein VP1C,which was found have a significantly better inhibitory effect on E-type Ct than VP1 protein.At the same time,the possible gene sequences of Ct-specific phage capsid protein VP1 were screened from the clinical samples of patients with Ct urogenital tract infection in the department of venereal diseases of Tianjin medical university general hospital,which showed a similarity of 97%-99%to the gene sequences of 6 kinds of chlamydia phage capsid protein VP1 discovered so far.[Objective]To design and construct a recombinant protein with high and specific inhibition effect against Ct and to explore a new solution for the treatment resistance of Ct infection.[Methods]E.coli containing recombinant plasmids 6His-VP1-pET30a(+)and 6His-VP1C-pET28a(+)were respectively induced to express a large number of VP1 protein and VP1C protein maintaining structure and function;The recombinant proteins were purified by His.Bind affinity resin with Ni~+,renatured by gradient dialysis,and identified by Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and Western blotting(WB)of the expression and purification;The purified VP1,VP1C protein and bacitracin were respectively preincubated with eight different serotypes of Ct(D,E,F,G,H,I,K,L1)standard strains for 3h at room temperature and then inoculated into Hela cells.Meanwhile,eight serotypes of Ct infection control groups were set up.The inclusion bodies were counted by indirect immunofluorescence staining after 48h;Compared the possible gene sequence of Ct-specific phage capsid protein VP1 screened from clinic samples with that of PhiCPG1 by BLAST alignment to identify the different gene locus.On the basis of the gene fragment of VP1C,to design the gene sequence of VP1Cm protein by changing the above different gene locus;The recombinant plasmid 6His-VP1Cm-pET28a(+)was constructed by gene synthesis and transformed into the E.coli BL21 competent cells,The recombinant VP1Cm protein gene were identified by antibiotic resistance plate screening,restriction enzyme digestion and nucleotide sequencing;The expression,purification and identification process of VP1Cm protein were as same as above;The protein concentration was measured by BCA protein quantification;The cytotoxicity of a series of diluted concentrations of VP1,VP1C,VP1Cm proteins and bacitracin on Hela cells was detected by CCK-8 method,and the optimal intervention concentration range in subsequent experiments was determined by the results;The same concentration of VP1,VP1C,VP1Cm protein,bacitracin and phosphate buffer solution(PBS)were respectively preincubated with E-type Ct standard strain at room temperature for 3hours and then inoculated into Hela cells,E-type Ct infection control group was set up at the same time.The inclusions were counted by immunofluorescence after 48 hours;The same concentration of VP1,VP1C,VP1Cm protein and bacitracin were respectively mixed with gonococcal solution,and uniformly coated to the chocolate plate.To count the number of colonies after 48h and observe the effects of VP1,VP1C,VP1Cm protein and bacitracin on the growth of gonococci.[Results]At the same intervention concentration,functional domain truncated protein VP1C had a better inhibitory effect on eight different serotypes of Ct than PhiCPG1capsid protein VP1(all P<0.0001).The inhibition rates of protein VP1 on eight serotypes of Ct were 78.19%,76.20%,77.89%,62.55%,64.60%,85.10%,73.97%and72.14%,respectively.The inhibition rates of protein VP1C on eight serotypes of Ct were 94.63%,85.13%,86.21%,81.46%,75.20%,89.64%,79.69%and 74.91%,respectively.Bacitracin had no obvious inhibitory effect on eight different serotypes of Ct(all P>0.05);Based on the VP1C gene sequence,the VP1Cm gene sequence was successfully designed,and the VP1Cm protein was successfully expressed and purified;At the same concentration,the inhibition rates of protein VP1,VP1C and VP1Cm on E-type Ct were 76.20%,85.13%and 90.01%,respectively.The inhibition effect of protein VP1Cm on E-type Ct was better than that of VP1(P<0.0001)and VP1C(P<0.01);Bacitracin has obvious inhibitory effect on gonococci(P<0.0001),while protein VP1,VP1C and VP1Cm have no obvious inhibitory effect on gonococci(all P>0.05).[Conclusion]Compared with PhiCPG1 capsid protein VP1,functional domain truncated protein VP1C has a general better advantage in Ct inhibition;The designed protein VP1Cm has better inhibitory effect on E-type Ct than protein VP1 and VP1C;The inhibitory effect of protein VP1,VP1C and VP1Cm on Ct was specific. |
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