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Shuttle vector pSHK4 for escherichia coli and haemophilus parasuis, preparation method and application

A technology of Haemophilus suis and Escherichia coli, applied in the direction of using vectors to introduce foreign genetic material, recombinant DNA technology, etc., can solve the problem that resistance selection markers cannot play a role

Inactive Publication Date: 2012-05-23
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some resistance selection markers commonly used in E. coli do not work in members of the Pasteurellaceae family

Method used

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  • Shuttle vector pSHK4 for escherichia coli and haemophilus parasuis, preparation method and application
  • Shuttle vector pSHK4 for escherichia coli and haemophilus parasuis, preparation method and application
  • Shuttle vector pSHK4 for escherichia coli and haemophilus parasuis, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Isolation, identification and bacteriological characteristics of Haemophilus parasuis isolate YC0093

[0038] 1. The bacteriological characteristics of Haemophilus parasuis isolate YC0093

[0039] Haemophilus parasuis isolate YC0093 forms round, smooth and moist, colorless and transparent colonies with a diameter of 1-2mm after cultured on TSA solid medium at 37°C for 24-48 hours. Observation under the microscope after Gram staining of above-mentioned colony smear, the bacterium is Gram-negative small bacillus, has pleomorphism from single coccus to long, slender, even filamentous thalline.

[0040] Haemophilus parasuis YC0093 cross-streaked with Staphylococcus aureus on the sheep blood agar plate, and cultured at 37°C for 24-48 hours, showing a typical "satellite growth phenomenon", that is, the closer the colony to the Staphylococcus grows, the better it is. Staphylococci do not grow far away, and hemolysis does not occur around H. parasuis colonies on bloo...

Embodiment 2

[0046] Example 2 Preparation of the shuttle carrier of the present invention

[0047] 1. PCR amplification of each element of the shuttle vector

[0048] Using the DNA of the commercial vector pBluescript II SK (+) (purchased from Strategene, USA) as a template, PCR amplification with primers Kpn (5'-CGACTCACTATAGGGCGAATTGGG) and OLF (5'-TTTTGCTCAGCGCGCAATTAACCCTCACTA) yielded a 176bp product, which contained The DNA fragment of the multiple cloning site (MCS); using the same template, a 699bp DNA fragment (UC ori) was amplified with primers OLR (5'-GGGTTAATTGCGCGCTGAGCAAAAGGC) and UCR (5'-TCAACCGTGGCCATGGGTAGAAAAAGATCAAAGGATCTTCTTG). Using the plasmid pLOF / TAG (a gift from Paul R. Langford, providing a registration form for disclosure of genetic resources sources) as a template, primers KF5'-CTTTTCTACCCATGGCCACGGTTGATGAGAGCTTTG) and KR (5'-TGTCCGCAACATATGCCTTCAACTCAGCAAAAGTTCGATT) were used to amplify the product (kan) with a length of 1191 bp. Using the wild plasmid pYC93 (...

Embodiment 3

[0076] Example 3: Verification of the shuttle function of the shuttle vector pSHK4

[0077] 1. Verify the replication of pSHK4 in E. coli:

[0078] The purpose of the present invention is to obtain a shuttle vector capable of replicating and expressing resistance markers in common cloning Escherichia coli engineering strains and Haemophilus parasuis. In the process of constructing the shuttle vector, it has been verified that the shuttle vector can replicate in Escherichia coli strain DH5α and make the host bacteria acquire kanamycin resistance. Using the calcium ion-mediated transformation method (Sambrook J., Russell DW., 2001. Molecular Cloning: A Laboratory Manual, third ed. Cold Spring Harbor Press, New York.), the shuttle vector pSHK4 obtained in the present invention was introduced into two other strains Escherichia coli strains HB101 (purchased from Invitrogen, USA) and Top10 (purchased from Invitrogen, USA) can also efficiently and stably obtain kanamycin-resistant p...

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Abstract

The invention relates to the technical field of genetic engineering and aims to construct a shuttle vector between escherichia coli and haemophilus parasuis. The shuttle vector is named as pSHK4 and comprises a nucleotide sequence expressed by a sequence table SEQ ID NO:1, wherein a coding area of the pSHK4 is located at the sequence corresponding to 927-1742 bit sequence of the nucleotide sequence and 271 amino acids are coded. The shuttle vector provided by the invention has the advantages that a duplicated starting locus of natural plasmids in a bacillus separating strain of the haemophilus parasuis is utilized to construct the shuttle vector suitable for the haemophilus parasuis for the first time, the shuttle vector has the characteristics of stable hereditary in pathogenic bacteria and can be used for performing genetic operation on the haemophilus parasuis, and the shuttle vector is a basic tool for researching a toxic factor of the haemophilus parasuis and has a significance in revealing a pathogenic molecular pathopoiesia mechanism.

Description

technical field [0001] The invention belongs to the field of microbial genetic engineering, and specifically relates to a shuttle carrier between Escherichia coli and Haemophilus parasuis and its preparation method and application. Exploration of the molecular pathogenic mechanism of pathogens. Background technique [0002] Haemophilus parasuis (Haemophilus parasuis) belongs to the genus Haemophilus Pasteurellaceae, is an important opportunistic pathogenic bacteria of pigs, often parasitizes the upper respiratory tract of pigs, and the pathogenic strains can break through the upper respiratory tract barrier , causing pig arthritis, meningitis, multiple serositis, known as Glaser's disease. The disease often occurs in pigs with high health level and low immune response level. At present, its prevalence is increasing year by year at home and abroad, causing certain economic losses to the global pig industry. Therefore, the pathogen has attracted more and more attention from ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/74
Inventor 徐晓娟陈利苹陈焕春蔡旭旺吴东方郭凤娟王湘如
Owner HUAZHONG AGRI UNIV
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