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Staphylococcus aureus inactivated vaccine for cow mastitis and preparation method thereof

A technology for dairy cow mastitis and staphylococcus, applied in vaccines, veterinary vaccines, antibacterial drugs, etc., can solve the problems of limited immune protection efficacy and antigen loss of inactivated whole bacteria vaccines

Active Publication Date: 2019-01-01
INNER MONGOLIA HUAXI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing studies have shown that inactivated whole-bacteria vaccines are better than subunit vaccines such as cracking and capsular polysaccharides, but the immune protection effect of inactivated whole-bacteria vaccines is limited and has not yet met the requirements of clinical immune prevention and control. The loss of key protective protein antigens during culture is closely related to

Method used

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  • Staphylococcus aureus inactivated vaccine for cow mastitis and preparation method thereof
  • Staphylococcus aureus inactivated vaccine for cow mastitis and preparation method thereof
  • Staphylococcus aureus inactivated vaccine for cow mastitis and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0133] Example 1: Screening and preservation of inactivated bacterial vaccine strains and bacterial strains for testing

[0134] 1. Screening and determination of prevalent strains of Staphylococcus aureus infected with cow mastitis

[0135] Nearly 30,000 milk samples for clinical mastitis were collected from 90 medium and large dairy farms covering 30 provinces and cities in my country, including Inner Mongolia, Chongqing, Guangdong, Heilongjiang, Gansu, Hebei, Shandong, and Xinjiang, and were separated and tested. To screen and determine the dominant prevalent strains of Staphylococcus aureus in dairy cow mastitis. Specific steps are as follows:

[0136] 1. Collection of milk samples from dairy cows

[0137] Select dairy cows with clinical symptoms of mastitis (or suspected subclinical mastitis), first scrub the udders with warm water, then 0.2% bromogeramine, and finally wipe the nipples with 70% alcohol. Samplers wipe and disinfect their fingers at the same time. Squeez...

Embodiment 2

[0171] Embodiment 2: Construction of recombinant SEB protein Escherichia coli BL21 strain

[0172] 1. Optimization of SEB sequence

[0173] The Staphylococcus aureus enterotoxin B (SEB) gene sequence is optimized, the optimized SEB gene sequence is shown in sequence 1 in the sequence listing, and the amino acid sequence of the encoded SEB protein is shown in sequence 2 in the sequence listing. The specific steps are as follows: According to GenBank (AB479118.1) and the SEB gene sequence of the reported site-directed mutation human vaccine, obtain its spatial three-dimensional structure model to mutate two amino acids at the binding residue of SEB and MHCII molecule and one amino acid on the TCRβ chain to reduce the The virulence of SEB, its protein antigen conservation, immunogenicity and antigen neutralization epitope characteristics were preserved. Point mutations were carried out at the 46th, 90th and 95th SEB hydrophobic binding region, polar binding region and disulfide ...

Embodiment 3

[0180] Embodiment 3: the construction of recombinant H1α protein Escherichia coli BL21 strain

[0181] 1. Optimization of Hlα sequence

[0182] The Staphylococcus aureus alpha hemolysin (Hlα) gene sequence is optimized, the optimized Hlα gene sequence is shown in sequence 3 in the sequence listing, and the amino acid sequence of the encoded Hlα protein is shown in sequence 4 in the sequence listing. The specific steps are as follows: design the Hlα gene sequence according to GenBank (X55185.1) and published literature, first of all, in view of the strong toxicity of Hlα, the active site H35 is mutated, and leucine Leu is used instead of histidine His . At the same time, in order to increase the expression of soluble Hlα protein, the base codon of the Hlα gene sequence was optimized to Escherichia coli preferred codons with a length of 885 bp using vector NTI advance 11 software. The optimized Hlα base sequence was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

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Abstract

The invention discloses a staphylococcus aureus inactivated vaccine for bovine mastitis and a preparation method thereof. The method comprises the steps of firstly isolating and identifying a sample of bovine mastitis milk juice, screening one strain of 5, 8, and 336 type staphylococcus aureus with good immunogenicity and stable passage, and performing steps including fermental cultivation, inactivation and the like to obtain an antigen stock solution of inactivated thallus; at the same time, constructing an SEB protein expression strain and an Hlalpha protein expression strain, and performingsteps including induction culture, centrifugation, purification, inactivation and the like to obtain an antigen stock solution of recombinant protein SEB and Hlalpha; and finally, mixing the antigenstock solutions with adjuvants to prepare the staphylococcus aureus inactivation vaccine for bovine mastitis. The staphylococcus aureus inactivation vaccine for bovine mastitis has good safety and caneffectively prevent infection of the 5, 8, and 336 type staphylococcus aureus. The staphylococcus aureus inactivation vaccine for bovine mastitis is of great significance to prevent and control popularity and spreading of bovine mastitis.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and in particular relates to an inactivated vaccine of cow mastitis staphylococcus aureus and a preparation method thereof. Background technique [0002] Bovine mastitis is an inflammatory lesion of dairy cow mammary gland caused by pathogenic or opportunistic pathogenic microorganism infection or physical and chemical stimulation. Because of its complex etiology and repeated attacks, it is one of the three major diseases that affect the sustainable development of the global dairy industry, and it is also the disease that causes the heaviest economic losses in the dairy industry, especially in my country. Therefore, the scientific prevention and control of cow mastitis has become a major practical issue related to the national economy and people's livelihood. [0003] There are many and complex pathogenic factors of dairy cow mastitis, such as pathogenic microbial infection, feeding methods, hy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/085A61P31/04A61P15/14
CPCA61K39/085A61K2039/521A61K2039/552A61P15/14A61P31/04
Inventor 王欢李春和王林叶朱平丽刘少蓉高强王伟
Owner INNER MONGOLIA HUAXI BIOTECH
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