Mixture of poly-pneumococcal capsular polysaccharide-protein conjugates and preparation method of mixture

A technology of pneumococcus and capsular polysaccharide, which is applied in the direction of botany equipment and methods, biochemical equipment and methods, medical preparations of non-active ingredients, etc., can solve the problem of inability to reduce the cost of vaccine production, short retention time, and ineffectiveness Protect the body from bacterial invasion and other issues, and achieve the effects of low preparation cost, good immunogenicity, and easy purification

Active Publication Date: 2014-01-08
KANVAX BIOPHARM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But this type of vaccine also has the following defects: First, polysaccharides with repeated chemical structures are T cell-independent type 2 immunogens. Without the participation of T cells, they cannot induce immune memory effects and stimulate the body to produce IgM and IgG2 antibodies , these antibodies have a short retention time in the body and cannot effectively protect the body from bacterial invasion; second, this type of vaccine cannot induce an immune response to prevent diseases in infants younger than 2 years old, and this population is immune Phylogenetically imperfect, high-risk groups prone to infectious diseases
If the inclusion body is to be turned into a soluble protein, it needs to be obtained by denaturation and renaturation, but the yield of the soluble CRM197 protein obtained by this process is still very low, which cannot reduce the cost of vaccine production and realize cheap mass production

Method used

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Embodiment 1

[0047] Example 1: A mixture of multiple pneumococcal capsular polysaccharide-protein conjugates, comprising 13 pneumococcal capsular polysaccharide-protein conjugates and aluminum phosphate adjuvant, each pneumococcal capsular polysaccharide-protein conjugate The material is the combination of corresponding serotype pneumococcal capsular polysaccharides with the same protein carrier through covalent bonds, and the 13 kinds of pneumococcal capsular polysaccharides are derived from pneumococcal serotypes 1, 3, 4, 5, 6A, 6B , 7F, 9V, 14, 18C, 19A, 19F and 23F were purified and extracted from the bacterial culture fluid, and the protein carrier was the A chain protein of the diphtheria toxin mutant CRM197 obtained by gene recombinant Escherichia coli expression, the gene recombinant The nucleotide sequence of the A chain protein of diphtheria toxin mutant CRM197 is:

[0048] 1 GGCGCTGACG ATGTTGTTGA CTCCTCGAAA TCCTTTGTTA TGGAAAACTT CTCCTCCTAC

[0049] 61 CACGGCACGA AACCGGGCTA T...

Embodiment 2

[0058] Embodiment 2: Change the adjuvant in embodiment 1 to aluminum hydroxide.

[0059] The preparation method of the mixture of the multivariate pneumococcal capsular polysaccharide-protein conjugate described in Example 1 is as follows:

[0060] First, the preparation of the A-chain protein of the diphtheria toxin mutant CRM197

[0061] 1. Confirmation of amino acid sequence of A chain of diphtheria toxin mutant CRM197 protein

[0062] The diphtheria toxin mutant CRM197 protein A chain expressed by genetic recombination is composed of amino acid polypeptides from 1 to 193 in the amino acid sequence of diphtheria toxin. The complete gene sequence of CRM197 (HW71379) was obtained from GenBank, and its amino acid sequence is as follows:

[0063] 1 GADDVVDSSK SFVMENFSSY HGTKPGYVDS IQKGIQKPKS GTQGNYDDDW KEFYSTDNKY

[0064] 61 DAAGYSVDNE NPLSGKAGGV VKVTYPGLTK VLALKVDNAE TIKKELGLSL TEPLMEQVGT

[0065] 121 EEFIKRFGDG ASRVVLSLPF AEGSSSVEYI NNWEQAKALS VELEINFETR GKRGQDAMYE

[006...

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Abstract

The invention discloses a mixture of poly-pneumococcal capsular polysaccharide-protein conjugates. The mixture contains 13 pneumococcal capsular polysaccharide-protein conjugates and an immunity-enhancement adjuvant, wherein each pneumococcal capsular polysaccharide-protein conjugate is formed by combining corresponding serum-type pneumococcal capsular polysaccharide with a same protein carrier through a covalent bond; the 13 pneumococcal capsular polysaccharides are obtained by purifying and extracting 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F bacteria; the protein carrier is A chain of a diphtherin mutant CRM197 obtained from expression of genetic recombinant escherichia coli. Meanwhile, the invention discloses a preparation method of the mixture. The mixture, compared to conventional bacterial fermentation total-chain diphtherin mutant CRM197, is easy to purify, high in yield and low in cost; experiments prove that the mixture disclosed by the invention is immunogenic, and is applicable to clinical inoculation.

Description

Technical field: [0001] The invention relates to a mixture of 13 pneumococcal capsular polysaccharide-protein conjugates with immunogenicity and a preparation method thereof. Background technique: [0002] The main victims of pneumococcal disease are children, especially in developing countries, millions of children die of pneumonia and meningitis every year. Currently, vaccination is one of the effective ways to protect children from pneumonia bacteria. Chemically, pneumococci possess a cell-surface capsular polysaccharide layer whose function is to help the pathogen infect the host. Capsular polysaccharides can shield bacterial cell surface functional components from being recognized by the host immune system, prevent the complement system from being activated by bacterial surface proteins and phagocytosis by immune cells, and if bacteria are phagocytized, capsular polysaccharides can prevent bacteria from being killed. Capsular polysaccharides with different chemical st...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/116A61K47/48C12N15/31C12N15/70C07K14/195A61P31/04A61K47/64
Inventor 李建平
Owner KANVAX BIOPHARM
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