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Acinetobacter baumannii and subunit protein combined vaccine and preparation method thereof

A technology of Acinetobacter baumannii and protein vaccines, which is applied in the interdisciplinary fields of molecular biology and immunology, can solve the problems of small batch-to-batch differences, toxic and side effects, and single ingredients, and achieve fewer adverse reactions, lower incidence rates, and better preparation simple effect

Active Publication Date: 2016-05-18
XIANGYA HOSPITAL CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is difficult to standardize the vaccine dose during the production process due to the many and complex components contained in it; and the excessive content of lipopolysaccharide (endotoxin) in it can produce obvious toxic and side effects
Corresponding recombinant protein vaccines such as Bap (biofilm associated protein), OmpA (outermembrane protein A) and Ata (trimeric autotransporter protein trimer autotransporter protein) are all conserved gene expression products with high homology and cover Most of the Acinetobacter baumannii strains have shown good immune effect, and have the advantages of single component, good repeatability, small difference between product batches, and easy control of bacterial endotoxin content, etc., but the single-component vaccine may have insufficient immune protection , and easily put Acinetobacter baumannii under strong selection pressure to induce down-regulation of cell membrane target proteins, resulting in immune evasion and final failure of the vaccine

Method used

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  • Acinetobacter baumannii and subunit protein combined vaccine and preparation method thereof
  • Acinetobacter baumannii and subunit protein combined vaccine and preparation method thereof
  • Acinetobacter baumannii and subunit protein combined vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Recombinant plasmid construction

[0048] (1) Design and synthesize SmpA and PLD sequence primers according to the SmpA and PLD gene sequences, introduce NdeI and XhoI restriction sites at the two ends of the primers, and synthesize SmpA and PLD genes by PCR;

[0049] (2) The PCR product was recovered and purified, TA cloned into the vector pMD18-T (purchased from Shanghai Haoran Biotechnology Co., Ltd.), and the cloning vector pMD18-SmpA and pMD18-PLD were constructed and transformed into E. coli BL21. The PCR screening was positive For transformants, a small amount of plasmids were extracted, and then identified and sequenced by restriction enzyme digestion;

[0050] (3) The correct pMD18-SmpA and pMD18-PLD were digested and sequenced with NdeI and XhoI, and the pET28b plasmid (purchased from Shanghai Beinuo Biotechnology Co., Ltd.) was ligated with T4DNA ligase to construct the expression plasmid pET28b. -SmpA and pET28b-PLD, and transform them into the expressi...

Embodiment 2

[0051] Example 2: Prokaryotic expression and purification of fusion protein ( figure 1 )

[0052] (1) Take 1ul of the recombinant pET28b plasmid to transform BL21(DE3), heat shock at 42°C for 90s, then stand on ice for 2min, then spread the plate (30ug / mL kanamycin), and incubate at 37°C overnight;

[0053] (2) Pick a single colony of the expression strain BL21(DE3) in a test tube (4mL LB medium, 30ug / mL kanamycin) 37°C, 220rpm overnight culture;

[0054] (3) Inoculate the cultured bacteria liquid in 4ml LB medium at a volume ratio of 1:100, add 30ug / mL kanamycin, and cultivate at 37°C and 220rpm;

[0055] (4) When the OD value reaches about 0.6, add IPTG with a final concentration of 0.2 mM, 20°C overnight, 37°C, 220 rpm for 5 hours, and no IPTG inducer is added as a negative control;

[0056] (5) Collect the bacterial cells and suspend them in PBS buffer;

[0057] (6) SDS-PAGE electrophoresis detection ( figure 1 A, D);

[0058] (7) Mass induction: Inoculate the bacterial solution cultu...

Embodiment 3

[0068] Example 3: SmpA and PLD are respectively immunized in animals to select appropriate doses

[0069] Recombinant SmpA and PLD were mixed with 1% aluminum hydroxide solution at a mass ratio of 5 / 25 / 125μg each at a mass ratio of 1:1, and the mixed solution was used for 3 subcutaneous injections on day 0, day 7, and day 21. Diabetic mice are kept in an SPF environment. Two weeks after the last immunization, blood was collected for antibody level ELISA. The antigen was coated with 10μg of recombinant SmpA and PLD during ELISA. The test results confirmed that the antibody titer reached 10 3 -10 6 , Confirmed the production of high titer specific antibodies, and selected SmpA25μg and PLD5μg as the vaccine dose ( figure 2 ).

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Abstract

The invention provides an acinetobacter baumannii and subunit protein combined vaccine and a preparation method thereof. According to the preparation method, acinetobacter baumannii SmpA (small protein A) represented as SEQ ID No.1 and an acinetobacter baumannii PLD (phospholipase D) nucleotide sequence represented as SEQ ID No.2 are connected to expression vectors respectively, SmpA and PLD recombinant plasmids are established respectively, recombinant SmpA and PLD proteins are subjected to induced expression respectively, and then the two proteins are mixed with adjuvants for preparation of the vaccine. Based on immunoinformatics and reverse genetics, antigenicity and immunogenicity of outer membrane protein components of the multi-drug-resistant acinetobacter baumannii are analyzed, gene cloning, protein expression, purification and immunological research are performed, the effective outer membrane protein components are screened out and combined, the safety and the effectiveness of the vaccine are improved, and the vaccine has more realistic and more attractive application prospect compared with conventional vaccines.

Description

Technical field [0001] The invention belongs to the cross-field of molecular biology and immunology, and specifically is an Acinetobacter baumannii combined subunit protein vaccine and a preparation method thereof. Background technique [0002] Acinetobacter baumannii exists in the hospital environment and colonizes multiple parts of the patient, causing hospital-acquired pneumonia, bloodstream infection, abdominal cavity infection, meningitis, skin and soft tissue infections, and urinary tract infections. Due to its strong ability to acquire drug resistance and clonal dissemination, Acinetobacter baumannii has become a global epidemic and a common pathogen with high drug resistance. In my country, Acinetobacter baumannii has become one of the most common pathogens of nosocomial infections. In 2013, China’s CHINET bacterial resistance monitoring showed that the detection rate of Acinetobacter baumannii was 11.97%, second only to Escherichia coli and pneumonia. Klebsiella, most of...

Claims

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Application Information

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IPC IPC(8): A61K39/104A61P31/04C12N15/70
CPCA61K39/104A61K2039/545A61K2039/55505C12N15/70C12N2800/101
Inventor 潘频华李海涛
Owner XIANGYA HOSPITAL CENT SOUTH UNIV
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