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Application of fish brms1a and ivns1a genes

A kind of technology of gene and semi-smooth tongue sole, applied in the field of molecular biology, can solve the problems such as no reports yet, and achieve the effect of improving antibacterial ability

Inactive Publication Date: 2019-11-12
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Current studies on Brms1a and Ivns1a are mainly focused on mammals, and their roles have not been reported in fish

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Construction of fish brms1a and Ivns1a gene expression plasmids pCNbrms and pCNIvns:

[0017] The sequences of fish brms1a and Ivns1a genes have been reported (GenBank accession number: EU519228 and DQ535486). The construction of pCNbrms is as follows: the cDNA of tongue sole was used as template, and the brms1a gene was amplified by PCR with primers F1 and R1. The PCR conditions were: 94°C 60s pre-denatured template DNA, then 94°C 40s, 60°C 60s, 72°C 60s, 5 cycles, then changed to 94°C 40s, 64°C 60s, 72°C 60s, 30 cycles. The PCR product was purified with the Tiangen DNA Product Purification Kit and ligated with the PCR cloning vector pBS-T (purchased from "Tiangen Biochemical Technology Co., Ltd.", Beijing) at room temperature for 2-4 hours, and the ligation mixture was transformed into Escherichia coli DH5α After containing 100 μg / ml anka penicillin (Ap), 40 μg / ml 5-bromo-4-chloro-3-indole-β-D-lactoside and 24 μg / ml isopropyl-β-D-thiogalactoside Glycosides were cult...

Embodiment 2

[0021] Application of Fish brms1a and Ivns1a Gene Expression Plasmids pCNbrms and pCNIvns

[0022] Step 1) Plasmid injection

[0023] Dilute the pCNbrms and pCNIvns of the above-mentioned Example 1 to 100 ug / ml in PBS, which is the dilution of pCNbrms and pCNIvns. Thirty zebrafish (weighing about 3.2 g) were randomly divided into 3 groups, 10 in each group. Name the 3 groups A, B and C respectively. Each fish in group A was injected with 100ul pCNbrms dilution, each fish in group B was injected with 100ulpCNIvns dilution, and each fish in group C (control group) was injected with 100ul PBS.

[0024] The composition of the PBS is by weight percentage: 0.8% NaCl, 0.02% KCl, 0.358% Na 2 HPO 4 .12H 2 O, 0.024% NaH 2 PO 4 , and the balance is water.

[0025] Step 2) bacterial suspension preparation: cultivate Edwardsiella tarda TX1 to OD in LB medium 600 to 1, then centrifuge (5000g, 4°C) for 10min. Collect the cells and suspend them in PBS to a final concentration of 1x1...

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Abstract

The invention relates to the field of molecular biology, in particular to application of fish genes brmsla and Ivnsla, application of the fish gene brmsla or Ivnsla in preparing preparations for resisting bacterial infection, and application of plasmids pCNbrms and pCNIvns for expressing the fish genes brmsla and Ivnsla in resisting bacterial infection. After fish is injected with the expressing plasmids of the fish genes brmsla or Ivnsla, the bacterial infection resisting capability of fish can be remarkably improved.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to the application of fish brms1a and Ivns1a genes. Background technique [0002] Brms1a (breast cancer metastasis suppressor 1a) and Ivns1a (influenzavirus NS1A binding protein a) are two novel genes that have been shown to have pro-apoptotic effects in mammals. Brms1a belongs to a class of tumor metastasis suppressor genes, which were first discovered in breast cancer. Brms1a can promote cell apoptosis and block the metastasis of various cancer cells by inhibiting the expression of osteopontin. Ivns1a is involved in the regulation of multiple cellular functions, including inhibition of nucleic acid transport, binding to the poly(A) sequence of mRNA, inhibition of splicing of pre-mRNA, and induction of cell apoptosis. The current research on Brms1a and Ivns1a is mainly concentrated in mammals, and its role in fish has not been reported yet. Contents of the invention [0003] T...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00A61P31/04
CPCA61K48/00
Inventor 孙黎周泽军
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI