Construction and application of A19 Brucella GroES deleted mutant strain

A technology of Brucella and mutant strains, which is applied in the field of Brucella vaccine research, can solve the problems that it is difficult to distinguish natural infection vaccines from immunizing animals, and that pregnant animals and humans cannot be used

Pending Publication Date: 2019-06-25
SHENYANG AGRI UNIV
View PDF2 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing Brucella bovine A19 vaccine still has strong virulence, can not be used for pregnant animals and humans, has no specific missing markers, and various serum reactions and...

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Construction and application of A19 Brucella GroES deleted mutant strain
  • Construction and application of A19 Brucella GroES deleted mutant strain
  • Construction and application of A19 Brucella GroES deleted mutant strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1A19

[0054] The construction method of embodiment 1.A19 Brucella GroES gene deletion mutant strain, comprises the following steps:

[0055] Step 1 PCR primer design

[0056] According to the whole genome sequence of A19 Brucella strain, two pairs of specific PCR primers GroESKan-N-F / GroESKan-N-R and GroESKan-C-F / GroESKan-C-R were designed on the upstream and downstream of the GroES gene, and the forward primers GroESKan-N-F and Recognition sites of restriction endonucleases BamH I and Sal I and their protective bases are added to the 5'-end of the reverse primer GroESKan-C-R of the downstream fragment respectively, and the reverse primer GroESKan-N-R of the upstream fragment and the downstream fragment The reverse complementary sequences of the upstream and downstream primers of the kana resistance gene were added to the 5'-ends of the forward primers GroESKan-C-F, respectively.

[0057] According to the pK19mobsacB vector sequence, a pair of specific primers GroESKan-F / GroESKan-R...

Embodiment 2

[0090] The virulence and immune protection of embodiment 2.A19 Brucella strain GroES gene deletion mutant

[0091] 1. Toxicity determination of mutant strains

[0092] Take TSA (tryptose soy agar) medium (tryptone 17.0g, soytone 3.0g, glucose 2.5g, NaCl 5.0g, K 2 HP0 4 2.5g, agar 20.0g, distilled water 1000mL) A19 Brucella strain grown on and step 1 obtained the GroES gene deletion mutant colony, make 1×10 with PBS respectively 5 CFU bacterial suspension, and then 75 6-8 week female BALB / c mice were randomly divided into three groups, and each mouse was injected intraperitoneally with 0.2mL A19 Brucella strain and GroES gene mutant strain bacterial liquid respectively, and in another Each mouse in the group was injected with 0.2 mL of normal saline as a blank control. On the 8th, 15th, 28th, 42nd and 60th days after infection, 5 mice from each group were sacrificed, the spleen was removed under aseptic conditions, weighed, and then 5 mL of PBS homogenate was added, and 10-f...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention relates to construction and application of an A19 Brucella GroES deleted mutant strain and belongs to the field of Brucella vaccine research. By designing PCR primers of an upstream region, a downstream region and a kana resistance gene of GroES, amplifying homologous nucleotide fragments of two lengths of the upstream and the downstream of a target gene and the kana resistance gene,directionally connecting the homologous nucleotide fragments with the kana resistance gene by adopting a PCR amplification method, defective homologous nucleotide fragments of the target gene are obtained, and the obtained defective homologous nucleotide fragments of the target gene are connected into a cloning vector to obtain a recombinant vector containing the defective homologous nucleotide fragments of the target gene, the constructed recombinant vector containing the defective homologous nucleotide fragments of the target gene is transformed into Brucella A19, positive clones are screened to obtain the Brucella A19 attenuated vaccine mutant strain of the deleted target gene. The vaccine mutant strain is expected to be developed into a marked attenuated vaccine, and plays a positivepromoting role in controlling the epidemic and transmission of brucellosis and the healthy development of national economy.

Description

technical field [0001] The invention relates to the technical field of Brucella vaccine research, in particular to the construction and application of an A19 Brucella GroES deletion mutant. Background technique [0002] Brucellosis, also known as Mediterranean relaxation fever, Malta fever, wave fever or undulating fever, is a zoonotic systemic infectious disease caused by Brucella, which can infect animals, such as cattle, sheep, pigs, and dogs. and animals such as camels and deer. Its clinical features are long-term fever, hyperhidrosis, arthralgia, and hepatosplenomegaly. The chronic phase of the disease may cause damage to multiple organs and systems. [0003] Brucella is a Gram-negative non-motile bacterium, non-capsulated (smooth type with microcapsule), positive for catalase and oxidase, absolutely aerobic, capable of reducing nitrate, intracellular Parasitic, can survive in many kinds of domestic animals. There are six species of Brucella: Brucella malta (Brucell...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): A61K39/10A61P31/04C12N15/74C12N15/66C12N15/11G01N33/569
CPCY02A50/30
Inventor 陈泽良刘宝山韩小虎隋昀原张欢刘金玲沈国顺
Owner SHENYANG AGRI UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap