CN112501091A

CN112501091A - Vibrio harveyi cheA gene silencing cell strain and application thereof

Info

Publication number: CN112501091A
Application number: CN201910870928.8A
Authority: CN
Inventors: 徐晓津; 李慧耀; 鄢庆枇; 江兴龙; 黄力行; 祁欣; 赵玲敏
Original assignee: Jimei University
Current assignee: Jimei University
Priority date: 2019-09-16
Filing date: 2019-09-16
Publication date: 2021-03-16
Anticipated expiration: 2039-09-16
Also published as: CN112501091B

Abstract

The invention belongs to the technical field of biology, and particularly relates to a Vibrio harveyi cheA gene silencing cell strain, and a construction method and application thereof. The cell strain is Vibrio harveyi cheA-RNAi, is preserved in China center for type culture Collection in 2018, 12 months and 20 days, and has the preservation number of CCTCC NO: m2018913, the construction method comprises the following steps: 1) designing and synthesizing specific shRNA of the cheA gene of the vibrio harveyi; 2) annealing to form double-stranded RNA; 3) extracting pACYC184 plasmid and carrying out double enzyme digestion by using BamHI and SpHI enzymes; 4) connecting the double-stranded RNA with the recovered product, and carrying out heat shock transformation on the double-stranded RNA into escherichia coli DH-5 alpha; 5) extracting recombinant plasmid, and performing electric shock transformation to introduce the recombinant plasmid into Vibrio harveyi to prepare the Vibrio harveyi cheA gene silencing cell strain. The Vibrio harveyi cheA gene silencing cell strain can be used for preparing medicaments or vaccines for preventing and treating Vibrio harveyi. Experiments show that the vibrio harveyi cheA gene silencing strain can effectively reduce the adhesion capacity of vibrio harveyi and has important significance for preventing and treating fish diseases.

Description

Vibrio harveyi cheA gene silencing cell strain and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a Vibrio harveyi cheA gene silencing cell strain and application thereof.

Background

In recent years, large yellow croaker breeding is rapidly developed in coastal areas of China, such as Fujian, Zhejiang and Guangdong. However, as the scale of propagation increases, the frequency of various infectious diseases, particularly those caused by Vibrio harveyi, increases. Vibrio harveyi (Vibiro harveyi) is an important pathogenic bacterium for the condition, is a serious pathogen of marine organisms, and is an important pathogen causing death of cage-cultured Asian weever.

Antibiotics are the main means for treating bacterial diseases of aquatic animals at present, and have the defects of generating bacterial drug resistance, causing drug residues and the like along with the wide application of the antibiotics in treating the bacterial diseases. The resistance of the large yellow croaker is gradually reduced, the disease problem is gradually serious, and great loss is brought to the large yellow croaker breeding industry. Therefore, the research and development of biological medicines or vaccines have important significance for preventing and treating fish diseases.

Disclosure of Invention

The invention aims to solve the technical problem of providing a Vibrio harveyi cheA gene silencing cell strain and an application thereof.

The invention is realized by the following steps:

the invention firstly provides a Vibrio harveyi cheA gene silencing cell strain which is Vibrio harveyi cheA-RNAi, is preserved in China center for type culture collection in 2018, 12 months and 20 days, and has the preservation number of CCTCC NO: m2018913, deposit address: wuhan university in Wuhan, China.

The invention also provides application of the Vibrio harveyi cheA gene silencing cell strain, and the Vibrio harveyi cheA gene silencing cell strain is used for preparing a medicament or vaccine for preventing and treating Vibrio harveyi.

The invention has the following advantages: experiments show that the vibrio harveyi cheA gene silencing strain can effectively reduce the adhesion capacity of vibrio harveyi and has important significance for preventing and treating fish diseases.

Drawings

The invention will be further described with reference to the following examples with reference to the accompanying drawings.

FIG. 1 shows the results of adhesion experiments.

FIG. 2 shows the results of the motility test.

Detailed Description

Example 1 construction of Vibrio harveyi cheA Gene-silenced Strain

The construction method of the vibrio harveyi cheA gene silencing strain comprises the following steps:

1) designing and synthesizing specific shRNA of the cheA gene of the vibrio harveyi:

F:5'-GATCCGCACCAAGTGATCGGTATTCTTTCAAGAGAAGAATAC CGATCACTTGGTGCTTTTTTGCATG-3'

R:5'-CAAAAAAGCACCAAGTGATCGGTATTCTTCTCTTGAAAGAAT ACCGATCACTTGGTGCG-3'。

2) annealing the shRNA to form double-stranded RNA;

the annealing reaction system is as follows:

3) the pACYC184 plasmid was extracted and double digested with BamHI and SpHI enzymes, and the linearized plasmid was recovered by purification after agarose gel electrophoresis.

4) The double-stranded RNA in the step 2) is connected with the recovered product in the step 3) by using T4 ligase, and is transformed into Escherichia coli DH5 alpha in a heat shock mode. And (3) screening by using an LB medium plate containing 34mg/mL chloramphenicol, and sequencing and verifying the screened strain, wherein the sequence obtained by sequencing contains specific shRNA, namely the connection is successful.

The ligation reaction steps are as follows:

A. preparing a connecting reaction mixed solution according to the following system:

linear plasmid 2. mu.L

shRNA 5μL

SolutionI 7μL

B. The reaction was carried out at 16 ℃ for 3 hours and immediately thereafter used for the conversion experiment.

5) The joint experiment is carried out on the Escherichia coli SM10 and a wild strain Vibrio harveyi in the step 4), the acceptance ratio is 5:1 (introducing a recombinant vector into a strain is a difficulty in preparing a silent strain, and the ratio of the acceptor bacteria is controlled to be a proper ratio, so that the success rate of introduction can be improved. But the introduction process is not completely controllable, and a certain sporadic nature is also required for successful introduction, so the construction process of the silent strain can not be repeatedly realized) to prepare a Vibrio harveyi cheA gene silent strain, and simultaneously, the original pACYC184 plasmid is jointed and transformed into a Vibrio harveyi preparation control strain. According to the resistance of the chloramphenicol carried by the pACYC184 plasmid, a TCBS plate containing 34mg/mL chloramphenicol is used for screening the Vibrio harveyi carrying the pACYC184 plasmid, and the strain obtained by screening is determined to be the Vibrio harveyi by NCBI comparison after 16srRNA sequencing.

Example 2 adhesion test

Large yellow croaker mucus (20 μ L) was added uniformly to a glass slide (22mm × 22mm) and fixed with methanol at room temperature for 20 minutes. The bacterial suspension was adjusted to a final concentration of OD600 ═ 0.3 (3.0 × 108CFU/mL) using sterile PBS. 200 μ L of the bacterial suspension was spread evenly on a mucus-coated slide, incubated at 28 ℃ for 2 hours, and washed three times with PBS. The bacteria were fixed with 4% methanol for 30 min and stained with 1% crystal violet for 3 min. The slides were then examined under an optical microscope (x 1000), 20 microscope fields were selected, and bacteria were counted. Sterile PBS was used as negative control and control strain v. harveyi was used as positive control. Three trials were performed per group.

Results of adhesion experiments: the wild strain had an adhesive weight of 1081.22 cells/fieldofaw, and the silent strain was 309.20 cells/fieldofaw. As shown in fig. 1.

Example 3 motility test

Overnight cultured vibrio harveyi was adjusted to OD600 ═ 0.3 in LB, 1 μ Ι _ suspension was added dropwise to LB plates (0.3% agar) and the plates were incubated at 28 ℃ for 24 hours with the control strain v. harveyi used as a positive control. The colony diameter was measured. Three independent biological replicates were performed per group.

Motility results: the diameter of the colony of the wild strain is 22.17mm, and the diameter of the colony of the silent strain is 17.51 mm. As shown in fig. 2.

Example 4 chemotaxis experiments

A capillary tube with an inner diameter of 0.1mm was sealed at one end and filled with large yellow croaker mucus. The tube was immersed in a sterile syringe containing 2.5mL of the bacterial suspension (1.0X 109 CFU/mL). After 1 hour incubation at 28 ℃, the tube contents were inoculated onto LB plates to accurately determine the number of bacterial cells in the tubes. Chemotaxis of bacterial cells was assessed by comparing the number of bacteria in the tubes, the control strain v. harveyi was used as a positive control, and capillaries filled with no mucus buffer were used as a negative control. Three independent biological replicates were performed per group.

Chemotaxis experiment results: the wild strain chemotactic amount is 680685.93CFU/mL, and the silent strain chemotactic amount is 458257.57 CFU/mL.

As can be seen from examples 2-4, in the adhesion experiment results, the silencing strain capacity is reduced, the rest are functions related to adhesion, and the silencing strain capacity is also reduced, which indicates that the Vibrio harveyi cheA gene silencing strain of the invention can effectively reduce the adhesion capacity of Vibrio harveyi, and can be used for preparing medicines or vaccines for preventing and treating Vibrio harveyi.

Although specific embodiments of the invention have been described above, it will be understood by those skilled in the art that the specific embodiments described are illustrative only and are not limiting upon the scope of the invention, and that equivalent modifications and variations can be made by those skilled in the art without departing from the spirit of the invention, which is to be limited only by the appended claims.

Claims

1. A Vibrio harveyi cheA gene silencing cell strain is characterized in that: is Vibrio harveyi cheA-RNAi, which is preserved in China center for type culture Collection in 2018, 12 months and 20 days, and the preservation number is CCTCC NO: m2018913.

2. The use of the Vibrio harveyi cheA gene-silenced cell strain of claim 1, wherein: the Vibrio harveyi cheA gene silencing cell strain is used for preparing a medicament or a vaccine for preventing and treating Vibrio harveyi.