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Expression, Purification of Neisseria Gonorrhoeae Surface Protein a Recombinant Protein and Studies on Its Immunocompetence

Author: HuYaoHua
Tutor: HuSiHai
School: Nanhua University
Course: Pathogen Biology
Keywords: Neisseria gonorrhoeae Neisseria surface protein A LTB Recombinant proteins Immune activity
CLC: R392
Type: Master's thesis
Year: 2009
Downloads: 20
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Abstract

Objective: expressed in E.coli BL21 pET-30a/nspA, pET-30a/ltB, and pET-30a/ltB-nspA recombinant proteins, purified refolding, analysis and immunogenicity of the antigen. Nasogastric immune BALB / c mice, the detect it induced humoral immune (especially mucosal immune response) and cellular immune response level to provide experimental evidence for the development of new, efficient gonococcal protein vaccine. Method: 1. Transformed pET-30a/nspA, pET-30a/ltB, pET-30a/ltB-nspA prokaryotic recombinant plasmid, line-PCR and double digestion of recombinant plasmid. Recombinant expression, purification and identification. The induced recombinant pET-30a/nspA, pET-30a/ltB and pET-30a/ltB-nspA expressing recombinant protein in E.coli BL21, optimize IPTG concentration and expression time; Ni-NTA affinity chromatography purification of recombinant proteins ; by SDS-PAGE and Western blot analysis and identification of expression products. 3 Determination of the immunological activity of the recombinant protein. Dialysis refolding of purified recombinant protein, BCA Protein concentrations were determined in France to LTB-NspA, NspA, the recombinant proteins nasogastric immune 4w age BALB / c mice, while setting the recombinant proteins LTB and protein dissolving solution (Solution Buffer) control group, the first immunization day, and the subsequent immune 2w, 4w the last immunization 2w underwent tail vein blood collection, separation of the serum and PBS lavage mouse reproductive tract to collect the reproductive tract lavage fluid, indirect ELISA serum specific IgG levels NspA and reproductive tract lavage fluid level of specificity SIgA in NspA. After the last immunization 2w mouse spleen cells cultured at 37 ℃ 5% CO2 incubator, MTT assay was used to detect the proliferation of splenocytes culture supernatant of INF-γ and IL-4 were measured by ELISA in spleen lymphocytes level. Results: 1. PET30a/nspA, pET30a/ltB, pET30a/ltB-nspA prokaryotic expression vector into E. coli BL21 after induction with IPTG, the recombinant fusion protein, Western-blot analysis of the expression of the protein have a strong The antigen can be combined with a specific antibody of the anti-NspA titer reached 1:6400 above. Recombinant protein, experimental group NspA, the LTB-NspA mouse reproductive tract mucosal SIgA water was significantly higher than that in the control group LTB, protein the dissolved solution group (Solution Buffer) (P lt; 0.01); splenocyte proliferation determination , the protein vaccine the LTB-NspA group, and NspA of mice spleen lymphocytes by the specific antigen NspA stimulation, the stimulation index (1.63 ± 0.265) and (1.59 ± 0.254) was significantly higher than the LTB group (1.11 ± 0.183), protein the dissolved solution group (Solution Buffer) (1.08 ± 0.162) (P lt; 0.01), the experimental group NspA LTB-NspA no significant difference (P gt; 0.05). Protein vaccine immunized mice spleen lymphocytes by specific antigen NspA of stimulation, culture supernatants of IFN-γ were significantly increased, NspA group of (134.58 ± 14.17 pg / mL) (142.23 LTB-NspA group ± 21.25 pg / mL), and the the LTB group of (46.12 ± 5.61 pg / mL) and protein the dissolved solution group (6.13 ± 1.06 pg / mL) between the significant difference (P lt; 0.01), the experimental group NspA LTB- the NspA no significant difference (P gt; 0.05); between the two experimental groups of mice spleen lymphocytes culture supernatant IL-4 were significantly increased after by specific antigen NspA stimulus, the experimental group NspA (82.26 ± 7.42 pg / mL) and LTB-NspA (86.14 ± 8.24 pg / mL) was significantly higher than the the LTB group of (36.52 ± 2.34 pg / mL) and protein dissolving solution (Solution Buffer) (31.15 ± 1.32 pg / mL) control group, the difference was significant (P lt; 0.05); between the two groups NspA and LTB-NspA, the level of IL-4 was no significant difference (P gt; 0.05). Conclusion: 1. Prokaryotic expression recombinant pET-30a/nspA, pET-30a/ltB and pET-30a / LTB-nspA in E.coli BL21 successful expression; recombinant protein immunized mice via nasogastric induce a certain level of specific humoral and cellular immune the the experimental group the LTB-NspA and NspA induced IgG, IL-4 and IFN-γ levels and spleen lymphocyte proliferative stimulation index, was significantly higher than that in the control group; 3. containing mucosa specificity the mucosal adjuvant the LTB-NspA of the induced the SIgA level significantly higher than the non-mucosal adjuvant NspA; but both induced IgG, IL-4, IFN-γ levels and spleen lymphocyte proliferation stimulation index difference no significant.

Expression, Purification of Neisseria Gonorrhoeae Surface Protein a Recombinant Protein and Studies on Its Immunocompetence

Abstract

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