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Shuttle plasmid and construction method and application thereof

A technology for shuttle plasmids and plasmids, applied in the biological field, can solve the problems of low conjugation transfer efficiency, large plasmid size, inability to stably exist Riemerella anatipestifer, and achieve the effect of high conjugation transfer efficiency and easy operation.

Inactive Publication Date: 2016-05-11
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently available shuttle plasmids are unstable, large in size, and low in conjugative transfer efficiency
The currently used shuttle plasmid is the shuttle plasmid pMM47.A of Capnocytophagacanimorsus (Capnocytophagacanimorsus), which cannot be stable in Riemerella anatipestifer due to the difference in the replication origin site of Riemerella anatipestifer exist

Method used

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  • Shuttle plasmid and construction method and application thereof
  • Shuttle plasmid and construction method and application thereof
  • Shuttle plasmid and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] According to the genetic background of Riemerella anatipestifer, a replication initiation gene capable of replicating in Riemerella anatipestifer is designed, and the specific sequence is shown in SEQ ID NO.1.

[0027] Then design the primers for amplifying SEQIDNO.1 according to the synthesized replication initiation gene sequence. For the convenience of vector construction, add HindⅢ and pstI restriction sites respectively at the upstream and downstream of the primers. The specific primers are as follows:

[0028] Upstream primer: RARepP15'-ccc aagctt gggctatttaggcattagccctc-5' (SEQ ID NO. 2);

[0029] There are primers below: RARepP25'-aaaa ctgcag ccaatgcattggaacagatctcgtatagagctcg-5' (SEQ ID NO. 3);

[0030] Using the synthesized SEQIDNO.1 as a template, SEQIDNO.2 and SEQIDNO.3 as primers for PCR amplification, the PCR amplification program was pre-denaturation at 98°C for 30s; denaturation at 98°C for 10s, annealing at 55°C for 30s, extension at 72°C for 1min30...

Embodiment 2

[0033] The shuttle plasmid pMY02 constructed in Example 1 was transformed into the donor strain Escherichia coli S17-1, and after 10 generations, the plasmid was extracted to detect the stability of the shuttle plasmid pMY02. The electrophoresis results were as follows: image 3 shown. The results showed that the structure of the shuttle plasmid pMY02 was stable after 10 passages, indicating that the shuttle plasmid pMY02 had good stability in Escherichia coli.

[0034] Then transfer the shuttle plasmid pMY02 to Riemerella anatipestifer RAATCC11845 by conjugative transfer method to obtain RAATCC11845::pMY02, then use 1 μg / ml of cefoxitin to screen positive clones, and then the screened bacterial strain RAATCC11845::pMY02 in the blood Upload 10 generations on the plate, and finally use SEQIDNO.2 and SEQIDNO.3 as primers for PCR detection, the results are as follows Figure 4 shown. The results showed that the structure of the pMY02 recombinant plasmid was stable after 10 pass...

Embodiment 3

[0036] After constructing the shuttle plasmid pMY02, the Riemerella anatipestifer TonB1 gene sequence was inserted into the NcoI and XbaI restriction sites of the shuttle plasmid pMY02, and the primers for TonB1 gene amplification were as follows:

[0037] Upstream primer: TonB1CompP1: 5'-catg ccatggatgagccaaaccataaatac-3' (SEQ ID NO. 4);

[0038] Downstream primer: TonB1CompP2: 5'-ctag tctaga ttagaaagtgattttgtaagtgcctg-3' (SEQ ID NO. 5);

[0039] Using the Riemerella anatipestifer genome as a template, and the sequences shown in SEQIDNO.4 and SEQIDNO.5 as primers, the PCR amplification conditions were pre-denaturation at 98°C for 30s; denaturation at 98°C for 10s, annealing at 50°C for 30s, and extension at 72°C for 30s. Cycle 30 times; then extend at 72°C for 10 minutes; finally store at 16°C. The amplified products were subjected to agarose gel electrophoresis, and the results were as follows: Figure 5 shown. The results showed that TonB1 was amplified, and the band...

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Abstract

The invention discloses a shuttle plasmid and a construction method and application thereof. The shuttle plasmid includes a starting replicator shown as SEQ ID NO.1, can be replicated in riemerella anatipestifer and can stably exist in escherichia coli and the riemerella anatipestifer. Therefore, the shuttle plasmid can be used for anaplerosis of gene knockout mutants of the riemerella anatipestifer and provide a powerful tool for identification of gene functions of the riemerella anatipestifer.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a shuttle plasmid, and also relates to a construction method and application of the shuttle plasmid. Background technique [0002] Riemerella anatipestifer is a contagious disease caused by Riemerella anatipestifer (RA), also known as duck infectious serositis. It has become one of the most common bacterial diseases that endanger the duck industry in all countries in the world. The current prevention and control methods mainly include drug prevention and vaccine prevention. However, Riemerella anatipestifer is resistant to multiple drugs, and it is difficult to find sensitive drugs for this bacteria in many areas. On the other hand, there are multiple serotypes of Riemerella anatipestifer, and there is no cross-protection among the serotypes. Therefore, the prevention and control effect of vaccines is not satisfactory. In-depth study of the pathogenic mechanism of the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/74C12N15/66
CPCC12N15/70C12N15/66C12N15/74C12N2800/101
Inventor 刘马峰王梦怡黄觅程安春汪铭书贾仁勇朱德康陈舜杨乔吴英孙昆峰
Owner SICHUAN AGRI UNIV
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