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Streptococcus pneumonia fusion protein and vaccine thereof

A Streptococcus pneumoniae and fusion protein technology, applied in the field of Streptococcus pneumoniae fusion protein and its vaccine, can solve the problems of poor immune effect, complicated coupling technology, high vaccine price, etc.

Inactive Publication Date: 2016-09-28
CHANGCHUN BCHT BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, neither the capsular polysaccharide vaccine nor the capsular polysaccharide-protein conjugate vaccine can cover more than 90 serotypes
The 23-valent capsular polysaccharide vaccine is not dependent on T cells, and cannot cause immune memory of immune cells after immunization, and the immune effect on infants whose immune system is not fully developed is poor, but infants are susceptible to Streptococcus pneumoniae infection The population, the vaccine is mainly used in adults; the Streptococcus pneumoniae polysaccharide-protein conjugate vaccine couples the capsular polysaccharide to the protein carrier, and after immunizing the body, it can cause a T cell-dependent immune response against the polysaccharide and induce immune memory , but each capsular polysaccharide needs to be coupled to a protein carrier. Due to the complexity of the coupling technology, the price of the vaccine is high, and it is difficult to be widely used in the world
However, there are safety issues with bacterial toxins, and it has been reported that cholera toxins can cause inflammation in the brains of mice

Method used

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  • Streptococcus pneumonia fusion protein and vaccine thereof
  • Streptococcus pneumonia fusion protein and vaccine thereof
  • Streptococcus pneumonia fusion protein and vaccine thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] Embodiment 1: Obtaining of nucleic acid sequence encoding target protein

[0101] 1. Selection of amino acid sequence of Plym2-PA fusion protein, acquisition of coding nucleotide sequence

[0102] PA is the 219-437 amino acid sequence of Lactococcus lactis cell wall hydrolase ACMA (GenBank: U17696.1), as shown in SEQ ID NO:1. Select codons favored by Escherichia coli to optimize the nucleotide sequence encoding PA amino acid sequence, introduce BamH I restriction site and linker sequence at the 5' end of nucleic acid synthesis, and introduce stop codon and Xho I at the 3' end restriction site, thereby obtaining the nucleotide sequence encoding the PA protein, as shown in SEQ ID NO:2. The coding nucleotide sequence of PA was entrusted to Shanghai Sangon Bioengineering Co., Ltd. for gene synthesis, and the synthetic PA coding nucleotide sequence was inserted into the pGH plasmid. It was sequenced and verified to be correct.

[0103] Plym2 is a mutant of Ply (pneumolysi...

Embodiment 2

[0147] Embodiment 2: the expression of fusion protein and the preparation of vaccine

[0148] 1. Construction of expression vector

[0149] The plasmid with the correct sequenced Plym2-encoded nucleic acid (SEQ ID NO: 6) was digested with Nde I and BamH I, and the Plym2 nucleic acid fragment was reclaimed; the sequence-corrected PA-encoded nucleic acid (SEQ ID NO: 2) The plasmid was digested with BamH I and Xho I to recover the PA nucleic acid fragment; the expression vector PET20b (purchased from Novagen) was digested with Nde I and Xho I to recover the vector fragment. The Plym2 fragment obtained by digestion with Nde I / BamH I, the PA fragment obtained by digestion with BamHI / Xho I, and the pET20b empty vector with sticky ends after digestion with Nde I / Xho I were co-ligated, and then transformed into E.coli Top10 (purchased from Tiangen Biochemical Technology Co., Ltd.), was added to the LB medium plate containing 50 μg / ml ampicillin and cultivated overnight, and the next da...

Embodiment 3

[0164] Embodiment 3: the immunogenicity detection of vaccine

[0165] BALB / c mice (16-18g) were divided into random groups, 6 mice in each group. Plym2-PA-BLP, PspA2-PA-BLP, PspA4-PA-BLP, PsaA-PA-BLP, PcpA-PA-BLP and PhtD-PA-BLP dissolved in PBS were used for nasal cavity immunization of mice, according to BLP The amount of BLP was calculated as 0.3 mg / mouse, and nasal cavity immunization was carried out 3 times; the negative control was PBS plus BLP, and the amount of BLP was calculated as 0.3 mg / mouse, and nasal cavity mucosal immunization was also carried out 3 times; the interval between each immunization was 14 days. Subcutaneous immunization control group is Plym2, PspA2, PspA4, PsaA, PcpA or PhtD protein purified by our laboratory, 20 μg protein per mouse and 100 μg Al(OH) 3 The adjuvant was mixed evenly, and the mice were immunized with the above mixture at three subcutaneous spots on the back respectively, for a total of 3 times with an interval of 14 days between ea...

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Abstract

The invention relates to the technical field of biology, in particular to streptococcus pneumonia fusion protein and a vaccine thereof. The fusion protein is an expression product formed by recombination of a streptococcus pneumonia virulence protein gene and a protein connector gene, and streptococcus pneumonia virulence protein is Ply or PspA or PsaA or PcpA or PhtD. After the vaccine of the streptococcus pneumonia fusion protein immunizes an animal through the nasal cavity, a high-potency serum specific IgG antibody can be induced, high-potency local mucosa specific IgA can be induced, and a good protective effect is achieved for nasal cavity toxin counteracting of different streptococcus pneumonia strains.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a Streptococcus pneumoniae fusion protein and a vaccine thereof. Background technique [0002] Streptococcus pneumoniae (Streptococcus pneumoniae) is an encapsulated Gram-positive bacterium that usually colonizes the oral cavity and nasopharynx of healthy people, and is one of the normal flora; when the human body's immunity is low, Local infiltration of Streptococcus pneumoniae can cause sinusitis and otitis media, inhalation into the lungs can cause pneumonia, and it can also invade the body and cause diseases such as bacteremia and meningitis. WHO estimates that about 1.6 million people worldwide die from pneumococcal disease every year, of which about 1 million are children under the age of five, accounting for 11% of the total mortality in this age group. [0003] The Streptococcus pneumoniae vaccines currently on the market are vaccines based on the Streptococcus pneumoniae ca...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21A61K39/09A61K39/39A61P31/04A61P37/04
CPCC07K14/3156A61K39/09A61K39/39A61K2039/55594C07K14/315C07K2319/00C12N15/70C12N2800/101C12N2800/22
Inventor 孔维吴永革卢井才王丹丹董运亮侯宏嘉谷铁军姜春来
Owner CHANGCHUN BCHT BIOTECH
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