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Application of calmodulin and calreticulin of bastard halibut

A technology of calreticulin and calmodulin, applied in the field of molecular biology, can solve problems such as unknown functions, and achieve the effect of improving antibacterial ability

Inactive Publication Date: 2015-07-15
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, fish calmodulin and calreticulin are rarely studied, and their functions are unknown

Method used

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  • Application of calmodulin and calreticulin of bastard halibut
  • Application of calmodulin and calreticulin of bastard halibut

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Construction of calmodulin and calreticulin expression plasmids pCNCam and pCNCrt:

[0016] The sequences of calmodulin and calreticulin from flounder have been reported (GenBank accession number: EU519228 and DQ535486). Calmodulin has a typical calcium ion binding domain (amino acids 85-147); calreticulin has a typical calreticulin domain (amino acids 20-331). The construction of pCNCam was as follows: the calmodulin gene was amplified by PCR using flounder cDNA as a template and primers F1 and R1. The PCR conditions were: 94°C for 60s to pre-denature template DNA, then 94°C for 40s, 63°C for 60s, 72°C for 60s, 5 cycles, then changed to 94°C for 40s, 65°C for 60s, 72°C for 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 c...

Embodiment 2

[0020] Application of Calmodulin and Calreticulin Expression Plasmids pCNCam and pCNCrt

[0021] Step 1) Plasmid injection

[0022] Dilute the pCNCam and pCNCrt of the above-mentioned Example 1 to 100 ug / ml in PBS, which is the dilution of pCNCam and pCNCrt. Thirty flounder (about 12 g in weight) 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 pCNCam dilution, each fish in group B was injected with 100ul pCNCrt dilution, and each fish in group C (control group) was injected with 100ul PBS.

[0023] 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.

[0024] Step 2) Bacterial suspension preparation

[0025] Cultivation of 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 ...

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Abstract

The invention relates to the field of molecular biology, specifically to preparation and application of expression plasmids of calmodulin and calreticulin of bastard halibut, specifically to application of the plasmids pCNCam and pCNCrt expressing calmodulin and calreticulin to antibacterial infection. When fish is injected with the expression plasmids of calmodulin and calreticulin, the fish has improved capability of resisting bacterial infection.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to the application of flounder calmodulin and calreticulin. Background technique [0002] Calcium is an indispensable ion for various physiological activities of the body, and plays an important role in maintaining cell membrane biopotential, normal nerve conduction, and muscle stretching and relaxation. Both calmodulin (CaM) and calreticulin (Crt) are Ca 2+ Binding protein that plays an important role in calcium metabolism. CaM is widely present in eukaryotic cells and can regulate intracellular Ca 2+ concentration; in the endoplasmic reticulum, CaM can also be used as Ca 2+ storage medium. The vital activities mediated by CaM include inflammatory response, apoptosis, muscle contraction, intracellular movement and immune response. Crt is able to bind Ca in the endoplasmic reticulum 2+ protein that maintains intracellular Ca 2+ steady state effect. Crt also acts as a chapero...

Claims

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Application Information

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IPC IPC(8): A61K38/17A61K48/00A61P31/04
Inventor 孙黎王晶晶
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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