A combined vaccine for the prevention of Staphylococcus aureus infection and tetanus

A staphylococcus, combined vaccine technology, applied in the fields of genetic engineering and immunology, can solve problems such as affecting the stability of the vaccine

Active Publication Date: 2020-12-22
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the dosage form of the combination vaccine, such as liquid or lyophilized powder, and their duration after mixing will affect the stability of the vaccine

Method used

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  • A combined vaccine for the prevention of Staphylococcus aureus infection and tetanus
  • A combined vaccine for the prevention of Staphylococcus aureus infection and tetanus
  • A combined vaccine for the prevention of Staphylococcus aureus infection and tetanus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1. Preparation of recombinant Staphylococcus aureus truncated SasA protein

[0027] For obtaining the recombinant Staphylococcus aureus truncated SasA protein, refer to Chinese patent application CN102268444A. The disclosure of CN102268444A is incorporated into the description of the present invention by reference. The preparation method is briefly described below.

[0028] 1. According to the reported sequence of Staphylococcus Aureus MASA252 strain (GenBank: BX571856.1), design primers to amplify the sequence of 142-999bp as the target fragment, and its amino acid sequence is shown in SEQ ID NO.2, which is the first part of the full-length protein 48-333 amino acid segment. NdeI and XhoI restriction sites were added to both ends of the SasA sequence, and the stop codon was removed, and a sequence encoding 6 histidines was introduced at the 3' end.

[0029]2. After the amplified SasA gene was digested with NdeI and XhoI, it was connected to the expression vect...

Embodiment 2

[0033] Embodiment 2. Preparation of recombinant tetanus neurotoxin C fragment (TeNT-Hc)

[0034] For obtaining the recombinant TeNT-Hc protein, refer to Chinese patent CN101880675B. The present invention incorporates the disclosure of CN101880675B into the description of the present invention in the form of reference, and the preparation method is briefly described below.

[0035] 1. According to the sequencing results (GeneBank sequence number: AF154828) of 64008 virulent strains of Clostridium tetani C.Tetani in China, the Tet-Hc sequence of 451Aa was analyzed and optimized, and the optimized sequence was shown in SEQ ID NO.3. Whole Gene Synthesis.

[0036] 2. Construction of Tetanus Toxin Subunit Vaccine Hc Expression Vector

[0037] After the optimized synthetic Tet-Hc gene was digested with EcoRI and XhoI, it was connected to the expression vector pET32a(+) which was also digested with EcoRI and XhoI, transformed into Escherichia coli competent cell DH5α, and cultured ov...

Embodiment 3

[0042] Example 3. Immunogenicity Research of SasA and TeNT-Hc Combined Vaccine

[0043] The following antigens were adsorbed with 0.75 mg aluminum hydroxide adjuvant: 10 μgrSasA, 10 μg TeNT-Hc, 10 μgrSasA+10 μg TeNT-Hc. 0.75 mg aluminum hydroxide adjuvant with no antigen adsorbed was used as negative control. At 0, 2 and 4 weeks, the above four groups of antigens were immunized with female BALB / c mice (aged 6-8 weeks) by intraperitoneal injection, 10 mice in each group. Blood samples were drawn from the tail vein and serum was extracted at 2, 4 and 6 weeks.

[0044] Serum antibody titers of antigen-specific IgG were analyzed by enzyme-linked immunosorbent assay (ELISA). 2 μg / ml of rSasA or TeNT-Hc was coated with 96-well enzyme-linked plate overnight at 4°C, and the coating buffer was 50 mM carbonate buffer (pH9.6). Blocking was incubated at 37° C. for 1 hour with PBS containing 2% (w / v) bovine serum albumin. The serially diluted serum was added to the enzyme-linked plate,...

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Abstract

The invention discloses a bivalent immunogen combined vaccine for preventing staphylococcus aureus infection and tetanus. The combined vaccine consists of recombinant staphylococcus aureus surface protein A (SasA) and tetanus neurotoxin C fragment (TeNT-Hc). The antibody response induced by the combined immunization of SasA and TeNT-Hc in a mouse is stronger than that of single component, and a synergistic effect is realized; and moreover, in a mouse staphylococcus aureus and tetanus toxin counteracting model, the combined vaccine shows relatively high protection property.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and immunology, more specifically, the invention relates to a method containing Staphylococcus aureus surface protein A (Staphylococcus aureus surface protein A, SasA) and tetanus neurotoxin C fragment (C fragment of tetanus neurotoxin, TeNT-Hc) bivalent immunogen composition and its application against Staphylococcus aureus and tetanus infection. Background technique [0002] Staphylococcus aureus and Bacillus tetani are widely distributed in nature, and any trauma patient with an open wound has the possibility of being infected with these two pathogenic bacteria. Staphylococcus aureus can cause skin and soft tissue infections, with fatal sepsis and invasive complications. Tetanus is caused by the obligate anaerobic bacterium Bacillus tetani, which produces neurotoxins that can attack the nervous system, causing generalized muscle spasms and even death by suffocation. Worldwide, tet...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/116A61K39/08A61K39/085A61P31/04
CPCA61K39/08A61K39/085A61K2039/70
Inventor 陈薇于蕊杨益隆候利华于长明徐俊杰郭强李建民付玲
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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