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a pneumococcal vaccine

A Streptococcus pneumoniae and vaccine technology, applied in the field of vaccines, can solve the problems of short-term immune response, low-affinity antibodies, and difficulty in immune enhancement, and achieve the effects of simple preparation, low cost, and good immune protection ability.

Active Publication Date: 2020-05-01
SINO UNITED (BEIJING) BIOMEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore polysaccharide vaccine has following problem: (1) can only produce weak immune response in young animal or infant body, even does not produce immune response, and immune response increases with age; (2) produces the antibody of low affinity; 3) It only produces a short-term immune response, and does not have the immune memory and immune enhancement effect during repeated vaccination; (4) It is easy to produce immune tolerance; (5) Common adjuvants are not easy to play an immune enhancement effect on this antigen
[0007] In PCT patent WO98 / 18930 published on May 7, 1998, titled "Streptococcus Pneumoniae antigens and vaccines" (Streptococcus Pneumoniae antigens and vaccines), many antigenic specific polypeptides are described, but for these polypeptides biological activity but no related reports

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Expression of Example 1 Protein

[0036]Using the DNA of Streptococcus pneumoniae as a template, the gene sequence of the amino acid sequence shown in SEQ ID NO: 1 was amplified by designing primers, and then the PCR was extracted from the agar gel using the QIAquick gel extraction kit of QIAgen (Chatworth, CA). Product recovery. After digestion with the Superlinker carrier pSL301 (Invitrogen, San Diego, CA), the QIAquick gel extraction kit of QIAgen (Chatworth, CA) was used to recover from the agar gel. The genomic DNA cut by the restriction enzyme The fragment was ligated with the pSL301 vector cut with restriction enzymes. The ligated product was then transformed into DH5a Escherichia coli according to the method of Simanis (Hanahan, D.DNA Cloning, 1985, D.M.Glover (ed), pp.109-13 5 Middle. The pSL301 recombinant plasmid (rpSL301) containing the target gene was purified with a QIAgen kit, and after sequencing, it was confirmed that the recombination was correct.

[...

Embodiment 2

[0038] The acquisition of embodiment 2 mutein

[0039] Introduction of mutation point

[0040] (1), design corresponding mutagenesis primers according to corresponding mutation sites, adopt PCR site-directed mutagenesis method to mutate the amino acid sites corresponding to the wild type on the corresponding S3CS gene; described mutation sites are in SEQ ID NO: 1 based on point mutations. F23V (indicates that the F amino acid at position 23 is replaced by V), K42C, P60T, K92S, P122D, E133F, D180S, F189H, K243S, I304G, L320Y, V329P, F332K, I339V, I352M, F361W, K369P, S372K, F380M , L401E.

[0041] (2), after the above-mentioned PCR product is recovered and purified by gel, it is digested with a restriction endonuclease, and after being ligated with the plasmid pSL301 carrier fragment through the same digestion, it is transformed into Escherichia coli DH5α competent cells;

[0042] (3) Identification of the recombinant plasmid: identification of the recombinant plasmid by enz...

Embodiment 3

[0044] Example 3 Antibody Effect Evaluation of Pneumococcal Protein Vaccine

[0045] 12-14g female 3-week-old BALB / c. young mice were randomly divided into 23 groups, 20 in each group, and were injected intraperitoneally with the conjugated vaccine prepared according to the proteins of Examples 1 and 2 and Al(OH)3 as an immune adjuvant, Commercially available 13-valent lung chain conjugate vaccine, blank control (PBS).

[0046] On the 0th, 2nd, and 4th weeks, the program injected 3 times, and each young mouse contained 0.5ml (0.5ug protein). Blood was collected from each experimental group 7 days after the third injection, the serum was separated, and stored at -20°C for future use.

[0047] For the detection of protein antibody titer, the indirect ELISA method was used respectively. The protein was dissolved in 0.05mol / L carbonate buffer solution with pH 9.6, coated with 20ug / ml concentration on the microtiter plate, and blocked with 2% BSA solution. During the experiment, ...

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Abstract

The invention discloses a streptococcus pneumoniae vaccine and a preparation method thereof. The streptococcus pneumoniae vaccine is prepared by mixing S3CS recombinant antigens or mutant protein antigens with additives. The vaccine can be applied to immunity; the protection of organisms on streptococcus pneumonia can be improved; a specific antibody can be generated. A preparation method of the vaccine is simple; the requirements of large-scale industrial production can be met.

Description

technical field [0001] The invention relates to vaccines, in particular to a Streptococcus pneumoniae vaccine. Background technique [0002] Infections caused by pneumococci (pulmonary chain) are a major cause of morbidity and mortality worldwide. Pneumonia, febrile bacteremia, and meningitis are the most common manifestations of invasive pneumococcal disease, while bacterial spread within the respiratory tract can lead to middle ear infection, sinusitis, or recurrent bronchitis. Noninvasive manifestations are usually less severe but more common than invasive disease. The likelihood of pneumococcal disease flares during influenza is further increased by the spread of antibiotic-resistant infectious diseases and the fact that pneumococcal pneumonia often follows influenza infection. Diseases caused by Streptococcus pneumoniae have become an important public health problem worldwide. Pneumococcus has become the number one killer of children worldwide. [0003] The case fat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/09A61K39/39A61P31/04A61P11/00C07K14/315C12N15/31
CPCA61K39/092A61K39/39A61K2039/6037A61K2039/6068A61K2039/6075C07K14/3156
Inventor 查文娟
Owner SINO UNITED (BEIJING) BIOMEDICAL TECH CO LTD
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