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Recombinant proteins PspA1, PspA2 and PspA3, and polysaccharide conjugation vaccine containing the same

A technology combining vaccines and recombinant proteins, applied in the field of recombinant proteins PspA1, PspA2 and PspA3 and polysaccharide conjugate vaccines containing them, can solve problems such as affecting immune effect, increasing tetanus toxoid inoculation, overloading or immunosuppression, etc.

Inactive Publication Date: 2018-02-09
NAT INST FOR FOOD & DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As the types of polysaccharide conjugate vaccines increase, the development of carrier proteins cannot keep up with the development of polysaccharide conjugate vaccines. It is bound to happen that the same carrier protein is used in several polysaccharide conjugate vaccines at the same time. If all polysaccharide conjugate vaccines use these proteins As a carrier, it will inevitably cause the phenomenon of competing for the limited carrier protein-specific T lymphocytes in the body, thereby affecting the immune effect
In addition, the component of tetanus vaccine is tetanus toxoid, which forms a combined vaccine with pertussis and diphtheria vaccines. It is already within the scope of EPI regulations. Repeated vaccination will inevitably increase the amount of tetanus toxoid inoculation, and it is currently used in combination with vaccines The content of the carrier protein is relatively large, and TT is the main carrier protein, which is likely to cause problems such as overload or immunosuppression[3,4]

Method used

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  • Recombinant proteins PspA1, PspA2 and PspA3, and polysaccharide conjugation vaccine containing the same
  • Recombinant proteins PspA1, PspA2 and PspA3, and polysaccharide conjugation vaccine containing the same
  • Recombinant proteins PspA1, PspA2 and PspA3, and polysaccharide conjugation vaccine containing the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1. Codon optimization and gene synthesis of the surface protein A gene sequence of Streptococcus pneumoniae

[0039] The gene sequence was synthesized by Zhongmeitaihe Biotechnology Co., Ltd., connected with the expression plasmid pET30a and transformed into E. coli expression strain BL21 (DE3). The gene sequence length is 1143bp for PspA1, 1122bp for PspA2, and 1431bp for PspA3. According to the gene sequence and amino acid sequence, the surface protein A of Streptococcus pneumoniae was artificially synthesized by means of codon optimization. The three branches synthesized were from strains DBL6A, RX1 and EF3296 (GenBank, sequence numbers were: AF071805.1, M74122.1 and AF071816), respectively, which were named PspA1, PspA2 and PspA3, and the signal peptide was removed, using The GENE designer software optimizes the codons of the selected gene sequence, and optimizes the rare codons into codons suitable for expression in the E. coli expression system to increas...

Embodiment 2

[0040] Example 2. Obtaining recombinant plasmids pET30a-PspA1, pET30a-PspA2 and pET30a-PspA3

[0041] The gene sequence was synthesized by Zhongmeitaihe Biotechnology Co., Ltd., and the target fragments of the three different branched proteins of PspA were cloned into the pET-30a(+) expression vector and transformed into the E. coli expression strain BL21(DE3). The transformed bacterial liquid was inoculated into LB liquid medium containing kanamycin, and the plasmid was extracted after shaking culture at 37°C for 12 hours.

[0042] In order to extract the plasmid, collect the precipitate of 1.5-3ml bacterial solution in a 1.5ml Eppendorf centrifuge tube, add 100μL of solution 1, shake until fully suspended. Add 150 μL of solution 2, and immediately invert the centrifuge tube gently several times to fully lyse the bacteria, and the lysed bacteria suspension becomes clear. The tubes were then placed on ice for 1-2 minutes. Add 150 μL of solution 3, immediately invert the cent...

Embodiment 3

[0051] Example 3. Induced expression of recombinant PspA1, PspA2 and PspA3 proteins in Escherichia coli BL21 (DE3)

[0052] Take 50 μL of the correct identified Escherichia coli BL21 (DE3) bacterial solution, inoculate it in 5 ml of LB liquid medium containing kanamycin (50 μg / ml), and cultivate overnight at 37° C. with shaking. Transfer to fresh LB liquid medium containing kanamycin (50 μg / ml) at a ratio of 1:50, culture at 37°C with shaking (200 rpm) until the 550nm OD value reaches 0.5-1.0. Add IPTG to a final concentration of 1 mM, and induce expression at 37°C for 4hs. Take 1ml of bacterial liquid, centrifuge at 5000rpm at 4°C for 5min, and collect the bacterial cells. The bacterial pellet was suspended in 100 μL 1xSDS loading buffer (50 mM Tris-Cl pH6.8, 100 mM DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol), and analyzed by SDS-PAGE electrophoresis.

[0053] According to "Molecular Cloning", prepare 12% separating gel and 5% stacking gel, cast the gel, and prepare e...

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Abstract

The present invention provides a nucleotide molecule, which has a sequence represented by SEQ ID NO.:2, 4 or 6. The present invention further provides a recombinant protein PspA1, PspA2 or PspA3 encoded by the nucleotide molecule of the present application. The present invention further provides a polysaccharide conjugation vaccine, which comprises meningococcal polysaccharide and a carrier protein bound to the meningococcal polysaccharide, wherein the carrier protein is the recombinant protein PspA1, PspA2 or PspA3 encoded by the nucleotide molecule of the present application. The present invention further provides uses of the recombinant proteins PspA1, PspA2 and PspA3 encoded by the nucleotide molecule of the present invention as the carrier protein of the polysaccharide conjugation vaccine. The present invention further provides an expression vector, which comprises the nucleotide molecule of the present invention.

Description

technical field [0001] This application relates to a method for synthesizing polysaccharide-bound vaccine carrier protein by artificially synthesizing gene sequences, more specifically, to a method for synthesizing pneumococcal surface protein A (Pneumococcal surface protein A, PspA) by artificially synthesizing gene sequences , and the method of combining it with meningococcal polysaccharide, and the clinical application of the polysaccharide combined with vaccine carrier protein. Background technique [0002] Bacteria with capsular polysaccharides, such as Haemophilus influenzae, Neisseria meningitidis, and Streptococcus pneumoniae, are several invasive respiratory bacteria that seriously threaten human health. Children and the elderly are high-risk groups for infection. Children under the age of 2, especially infants and young children, are the main fatal pathogens. Vaccination is an important means of preventing and controlling corresponding diseases. Bacterial polysac...

Claims

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Application Information

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IPC IPC(8): C07K14/315C12N15/31C12N15/70A61K39/385A61K39/09A61K39/095A61K47/62A61P31/04
CPCA61K39/092A61K39/095A61K39/385A61K2039/6087C07K14/3156A61K2300/00
Inventor 王丽婵谭亚军张庶民马霄侯启明卫辰张华捷
Owner NAT INST FOR FOOD & DRUG CONTROL
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