Milk cow Streptococcus uberis subunit vaccine GapC protein, and preparation method and application thereof

A subunit vaccine, cow mastitis technology, applied in the field of bioengineering, can solve the problem of not being able to significantly reduce mastitis, and achieve broad-spectrum protective effects

Inactive Publication Date: 2012-01-04
HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Giraudo et al. used multiple inactivated vaccines of Streptococcus uberis and Streptococcus dysgalactiae, and vaccination did not significantly reduce the occurrence of mastitis caused by Streptococcus in dairy cows

Method used

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  • Milk cow Streptococcus uberis subunit vaccine GapC protein, and preparation method and application thereof
  • Milk cow Streptococcus uberis subunit vaccine GapC protein, and preparation method and application thereof
  • Milk cow Streptococcus uberis subunit vaccine GapC protein, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The construction of embodiment 1 genetic engineering recombinant bacterium pQE-30-GapC / XL-1-Blue and the preparation of bovine streptococcus GapC protein subunit vaccine

[0029] (1) Preparation of the GapC gene of the bovine Streptococcus dysgalactiae subunit vaccine.

[0030] DNA extraction: Take 1.5 mL culture of Streptococcus dysgalactiae, such as LS0312 strain, centrifuge at 10,000 rpm for 2 min; add 567 μL TE to the precipitate, and pipette repeatedly. Then add 30 μl 10% SDS and 3 μl 20 mg / mL Proteinase K, mix well, and bathe in 37°C water for 1 hour; add 100 μl 5M NaCl, mix well, then add 80 μl CTAB / NaCl, mix well. Water bath at 65°C for 10 minutes; add an equal volume of chloroform / isoamyl alcohol, mix well, centrifuge at 12000rpm for 4-5min, transfer the supernatant to a new tube; add an equal volume of phenol / chloroform / isoamyl alcohol, mix well, and centrifuge at 12000rpm 5min, transfer the supernatant to a new tube; add 0.6 volume of isopropanol, mix lightl...

Embodiment 2

[0039] Example 2: Induced expression of recombinant bacteria pQE-30-GapC and purification of GapC protein

[0040] Inoculate 100 μL of pQE-30-GapC recombinant bacteria into 10 mL of LB medium containing 100 μg / mL ampicillin, culture at 37 ° C, when OD600 is about 0.5, aspirate 1 mL of the culture and place it in a 1.5 mL eppendorf tube. Add 100 mM inducer IPTG to a final concentration of 1 mM, and continue to culture with vigorous shaking for 6 h. Aspirate 1 mL of the bacterial solution every 1 h, centrifuge at 10,000 rpm, discard the supernatant, add 90 μL of 1× loading buffer and 10 mL of 1M DTT to the pellet, boil for 5 min, and take 15 μL for SDS-PAGE. see Figure 4 . The QIAexpressionistTM protein purification system was used for purification. Take 5 mL of the overnight cultured bacterial liquid in 0.5 L of LB liquid medium containing 50 μg / mL ampicillin, culture at 37°C for 3 h with shaking, add IPTG to a final concentration of 1 mmol / L and continue to culture for 4 h...

Embodiment 3

[0042] Embodiment 3: the fermentation culture condition optimization of engineering bacterium

[0043] (1) TR-GapC engineering bacteria

[0044] Preparation of Engineering Bacteria Seed Solution

[0045] Dissolve TR-GapC engineered bacteria, streak and inoculate them in LB solid medium containing Amp resistance, and culture at 37°C for 24 hours; then pick well-growing single colonies and inoculate them in 3 mL of LB liquid culture medium containing Amp resistance Incubate in a basal test tube at 37°C and 190rpm for 16 hours, use the sterilized basal seed medium as a blank control, measure the OD 600 of each bacterial solution, and when the OD 600 of the bacterial solution is between 1.3 and 1.5, it is regarded as a first-class seed. Take the first-grade seeds, inoculate them in LB liquid medium, and culture them at 37°C and 190rpm for 16 hours. After inspection, they are pure and stored at 2-8°C. They will be used as the second-grade seeds for experiments such as optimization...

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Abstract

The invention provides a milk cow Streptococcus uberis subunit vaccine GapC protein, and a preparation method and application thereof. The amino acid sequence of the GapC protein is disclosed as SEQ ID NO.1. Since the subunit vaccine prepared from bovine-derived streptococcus GapC protein has a cross protective function on three common milk cow Streptococci uberis, compared with other vaccines, the milk cow Streptococcus uberis subunit vaccine GapC protein provided by the invention has a more broad-spectrum protective function, and can be used as a candidate antigen of a milk cow uberis subunit vaccine.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a dairy cow Streptococcus mastitis subunit vaccine GapC protein and a preparation method and application thereof. Background technique [0002] Bovine mastitis (Bovine mastitis) is one of the major diseases that endanger the dairy cattle breeding industry in my country. According to the research of many scholars at home and abroad, Streptococcus is one of the most important pathogens causing mastitis. At present, domestic mastitis vaccines are mainly whole-bacteria inactivated vaccines. Although the vaccine has a certain protective power, it does not have the protective power of heterologous strains, so it does not have the effect of immune prevention against new bacteria. Mackie et al. reported that the use of formalin-inactivated Streptococcus agalactiae bacterin was not protective for dairy cows. Giraudo et al. used multiple inactivated vaccines of Streptococcus uber...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/315C12N15/31C12N15/63A61K39/09A61P31/04
Inventor 朱战波崔玉东车车张军于立权周玉龙柳强朱洪伟王鹤杨玉英
Owner HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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