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Main outer membrane protein epitope vaccine of chlamydia trachomatis based on HBcAg vector and application of main outer membrane protein epitope vaccine

A major outer membrane protein, Chlamydia trachomatis technology, applied in the fields of biomedical technology and immunology, can solve problems such as unsatisfactory effects

Inactive Publication Date: 2015-04-29
WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, research on Ct vaccines mainly includes whole bacterial vaccines, subunit vaccines, recombinant vaccines, and synthetic peptide vaccines, but the effects are not satisfactory.

Method used

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  • Main outer membrane protein epitope vaccine of chlamydia trachomatis based on HBcAg vector and application of main outer membrane protein epitope vaccine
  • Main outer membrane protein epitope vaccine of chlamydia trachomatis based on HBcAg vector and application of main outer membrane protein epitope vaccine
  • Main outer membrane protein epitope vaccine of chlamydia trachomatis based on HBcAg vector and application of main outer membrane protein epitope vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0105] Embodiment 1. Obtaining and modification of HBcAg carrier gene

[0106] Using the serum samples of hepatitis B virus carriers (Dasanyang) in Wenzhou area as a template, PCR primers were designed and synthesized, and enzyme cutting sites (Ned I and Hind III) were introduced at both ends to amplify the complete open reading frame of HBcAg. The PCR product and The vector pET21a(+) was ligated after double digestion at the same time to construct the recombinant plasmid pET21a(+) / HBcAg(wt), which was identified by sequencing.

[0107] In order to enhance the carrier effect of HBcAg and facilitate the operation of molecular cloning, the HBcAg nucleotide (SEQ ID NO: 3) was modified and designed, that is, the universal Th epitope PADRE was inserted into its C-terminus, and the N- and C-terminals of the HBcAg carrier Each introduces a His-tag sequence to facilitate purification; at the same time, base mutations are performed in its MIR region (78th and 82nd amino acids) to for...

Embodiment 2

[0108] Example 2. Preparation and in vitro expression of HBcAg / CtMOMP epitope vaccine, analysis of VLPs formation

[0109] Based on the prokaryotic codon-optimized Ct MOMP immunodominant epitope gene and tandem epitope gene, complementary primers were designed and synthesized, and the two ends were respectively introduced with restriction sites BamH I and Sac I, and the target gene was obtained after annealing. The annealed product was ligated with pET21a(+) / HBcAg digested by BamH I and Sac I to construct a recombinant prokaryotic expression plasmid containing the Ct MOMP epitope gene and the tandem epitope gene in the HBcAg MIR region; at the same time, the Ct MOMP Immunodominant epitopes were respectively connected to the N and C ends of the modified HBcAg, compared for research, and identified by sequencing ( figure 2 ). The results showed that: HBcAg(MIR) / Ct MOMP epitope 1, HBcAg(MIR) / Ct MOMP epitope 1+2, HBcAg(MIR) / CtMOMP epitope 1+2+3, Ct MOMP epitope 1 / HBcAg and HBc...

Embodiment 3

[0111] Example 3. Immunogenicity and immune protection effect of HBcAg / CtMOMP epitope vaccine

[0112]Female BALB / c mice aged 6-8 weeks (purchased from Shanghai Slack Experimental Animal Co., Ltd.) were randomly divided into 8 groups, 21 mice in each group, respectively: PBS control group, HBcAg group, HBcAg(MIR) / MOMP epitope 1 (MIR region carries dominant epitope 1), HBcAg(MIR) / MOMP epitope 1+2, HBcAg(MIR) / MOMP epitope 1+2+3(, Ct MOMP epitope 1 / HBcAg( N-terminal), HBcAg / Ct MOMP epitope 1 (C-terminal) and Ct MOMP epitope synthetic peptide group. Take 100 μl test samples (1tg / μl) at 0, 2, and 4 weeks respectively, and inject them into the back of the mouse for immunization Collect tail vein blood and vaginal secretions at 0w, 2w, 4w, 6w, 8w, 10w, 12w, 14w, 18w, and 20w after immunization, and detect Ct-specific IgG antibodies in serum and IgA antibodies in secretions by ELISA; In the 7th week, the spleen was taken to prepare splenocyte suspension, and the specific CTL killi...

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Abstract

The invention relates to a chimeric vaccine of a hepatitis B virus and chlamydia trachomatis, and an application of the chimeric vaccine, and particularly relates to a recombinant protein containing one or more epitopes with a main outer membrane protein epitope vaccine of the chlamydia trachomatis. The epitope peptides are inserted into the same or different permissive sites of hepatitis B virus core antigen (HBcAg) protein in a form of a single epitope or a plurality of epitopes which are combined together; the recombinant fusion protein capable of stimulating the organism to generate humoral immunity and cellular immunity with respect to the chlamydia trachomatis is formed; the immunogenicity and the immunoprotecive property of various fusion proteins are primarily evaluated; and a foundation is laid for further research and application of preventing and treating chlamydia trachomatis infection and related diseases employing the epitope vaccine based on an HBcAg vector. The vaccine disclosed by the invention has efficient and safe immunoprophylaxis and treatment effects on the related diseases of the chlamydia trachomatis, and is simple in preparation process and immunologic process, obvious in effect and high in repeatability.

Description

technical field [0001] The invention relates to the fields of biomedicine technology and immunology, in particular to a Chlamydia trachomatis main outer membrane protein immunodominant epitope vaccine with hepatitis B virus core antigen as a carrier, its preparation method, and its role in preventing and treating Chlamydia trachomatis infection in the application. Background technique [0002] Chlamydia trachomatis (Ct) is the most common pathogen of sexually transmitted diseases (STDs) in the world, which can cause urethritis in men and cervicitis in women. According to reports, 50-60% of urethritis in men is caused by Ct infection (Tiodorovi.J, Randelovi .G, Koci.B et al, Bacteriological finding in the urethra in men with and without non-gonococcal urethritis, 2007, 64(12): 833-6), and often mixed infection with other pathogens, sexually transmitted infection of female urinary Reproductive tract, leading to adverse pregnancy, such as miscarriage, stillbirth, etc., and is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/10C12N15/70C12N1/21A61K39/118A61P31/04
Inventor 张丽芳朱珊丽薛向阳李文姝陈韶陈俊林晓云
Owner WENZHOU MEDICAL UNIV
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