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Enhanced polysaccharide antigen immunogenic protein carrier as well as preparation method and application thereof

A polysaccharide antigen and protein carrier technology, applied in the field of polysaccharide protein conjugate vaccine development, can solve the problems of difficult quality control, large molecular weight of TT, residual toxicity of toxoid, etc., and achieve easy quality control, reduce immune interference, and enhance immunogenicity. Effect

Active Publication Date: 2017-02-22
CANSINO BIOLOGICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The conjugate vaccines marketed in China mainly use TT as the protein carrier. Although the conjugate vaccine developed with TT as the protein carrier shows a good immune effect, TT has a large molecular weight, strong allergenicity, and the coexistence of monomers and polymers. , the quality is difficult to control, and the toxoid after detoxification has the risk of residual toxicity and toxicity reversal. In addition, the single and repeated vaccination of the vector may affect the immunological effect of the vaccine, or cause immunosuppression induced by the vector

Method used

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  • Enhanced polysaccharide antigen immunogenic protein carrier as well as preparation method and application thereof
  • Enhanced polysaccharide antigen immunogenic protein carrier as well as preparation method and application thereof
  • Enhanced polysaccharide antigen immunogenic protein carrier as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Preparation of protein carrier CRM197A-Hin47

[0039] Step 1: Construction of CRM197A-Hin47 expression vector

[0040] Using the CRM197A and Hin47 gene sequences as templates and G4S as a linker, the target gene of the fusion protein can be amplified by over-lap PCR, such as figure 1 as shown, figure 1 It is the agarose gel electrophoresis image of over-lap PCR amplification product.

[0041] Both the fusion gene and the pET 9a expression vector were double-digested with BamHI restriction enzyme and NdeI restriction endonuclease, and then ligated by T4 DNA ligase, overnight at 16°C; the recombinant expression vector was transformed into DH5a competent, picked Single clone, inoculated in 5ml LB medium, cultured overnight at 37°C 200rpm; extract the plasmid for enzyme digestion identification, the verification results are as follows figure 2 as shown, figure 2 Indicates that CRM197A is successfully linked to the Hin47 gene.

[0042] The amino acid composit...

Embodiment 2

[0060] Example 2: Study on Immunogenicity of CRM197A-Hin47 Fusion Protein

[0061] Select 100 to SPF grade 10-12g female BALB / c mice for immunogenicity research, the method is as follows: the mice are randomly divided into 10 groups, 10 in each group. Abdominal subcutaneous immunization, the volume of immunization is 0.2mL, and the composition and content information of mice in each group are shown in Table 3. Immunization was carried out on the 0th and 14th day respectively. On the 28th day, the eyeballs were picked to collect whole blood, and the serum was collected by centrifugation at 8000rpm for 6 minutes, and stored at -20°C. The antibody titers against Hin47 and CRM197A proteins in mouse serum were determined by indirect ELISA.

[0062] Table 2 Components and contents of mice immunization in each group

[0063] Group No Sample serial number immune dose 1 Hin47-1 1μg 2 Hin47-2 5μg 3 Hin47-3 10μg 4 CRM197A-1 1μg 5 CRM1...

Embodiment 3

[0067] Example 3: Preparation of CRM197A-Hin47-PRP conjugate vaccine

[0068] Step 1: Covalent binding of Haemophilus influenzae type b capsular polysaccharide PRP to CRM197A-Hin47

[0069] Prepare Haemophilus influenzae type b capsular polysaccharide with common laboratory method, get appropriate polysaccharide and be dissolved in purified water (10mg / ml), 1-cyano group-4-dimethylamino-pyridine tetrafluoroboric acid (1-Cyano -4-dimethylaminopyridiniumtetrafluoroborate, CDAP) was dissolved in acetonitrile (100mg / ml), and CDAP was added at a ratio of 0.75mg CDAP / mg to activate the reaction for 30s. Then add 0.2M TEA (10ul / mg), adjust the pH value to 9.5, add fusion protein CRM197A-Hin47 (5mg / ml) after 150s, react at room temperature for 1h, react overnight at 4°C, add 5 drops of 2M glycine solution to terminate the binding reaction. After diafiltration, separate and purify.

[0070] Step 2: Purification of CRM197A-Hin47-PRP

[0071] The conjugate was separated and purified o...

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Abstract

The invention discloses an enhanced polysaccharide antigen immunogenic protein carrier as well as a preparation method and application thereof. A CRM197A gene sequence and a Hin47 gene sequence are taken as templates, and a fusion gene is obtained by over-lap PCR (polymerase chain reaction) amplification; after being subjected to double-enzyme digestion of BamHI and NdeI, the fusion gene and a pET 9a expression carrier are connected by T4 ligase; after successful connection is confirmed by enzyme digestion, CRM197A-Hin47 fusion protein is obtained by means of IPTG inducible expression and purification. Haemophilus influenzae type b capsular polysaccharide PRP and the fusion protein are coupled by a CDAP / CNBr or reduction amine method, and a prepared conjugate of the PRP and the fusion protein can be used for preparing a conjugate vaccine for preventing haemophilus influenzae type b. Experiments prove that when the fusion protein is taken as a protein carrier of a polysaccharide conjugate vaccine, the immunogenicity of polysaccharide antigen can be remarkably enhanced; furthermore, vaccines which are developed based on the protein carrier provided by the invention can effectively reduce the immune interference caused by currently using a great deal of same protein carriers, so that the development of combined vaccines and polyvalent vaccines is facilitated.

Description

technical field [0001] The invention relates to the technical field of polysaccharide-protein conjugated vaccine development. Specifically, the present invention develops a novel conjugated vaccine protein carrier by constructing a CRM197A-Hin47 fusion protein, and further develops a novel polysaccharide-protein conjugated vaccine. Background technique [0002] CRM197 is an avirulent mutant of diphtheria toxin. It has been proved by research that its immunogenicity is almost the same as that of diphtheria toxin. It is a protein carrier commonly used in polysaccharide protein vaccines in the world in recent years. CRM197 consists of two subunits, A and B, of which the B subunit has no enzymatic activity, but can bind to specific receptors on the surface of host susceptible cells, and allow the A subunit to enter the cell through translocation. The wild-type diphtheria toxin A subunit has enzymatic activity and can hydrolyze oxidized nicotinamide adenine dinucleoside into nico...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70A61K39/102A61K39/39A61P31/04
CPCA61K39/102A61K39/39C12N15/70C07K14/285C07K14/34A61K2039/55544C12N2800/101
Inventor 李军强杨鸣鸣莫清珊雷二铭邱琳李曦廖正芳邵忠琦
Owner CANSINO BIOLOGICS INC
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