Trachinotus ovatus beta-thymosin and application thereof

A technology of oval pomfret and thymosin, which is applied in the field of molecular biology, can solve the problems of unclear function and application potential of beta-thymosin, and achieve the effect of improving disease resistance.

Active Publication Date: 2017-08-22
HAINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • Trachinotus ovatus beta-thymosin and application thereof
  • Trachinotus ovatus beta-thymosin and application thereof
  • Trachinotus ovatus beta-thymosin and application thereof

Examples

Experimental program
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Embodiment

[0024] A kind of oval pomfret β-thymosin, the cDNA nucleotide sequence of the oval pomfret β-thymosin is as SEQ ID NO.1; The amino acid sequence of the oval pomfret β-thymosin is as SEQ ID NO.1 ID NO.2.

[0025] Extract the RNA of the ovoid pomfret, reverse transcribe the RNA into cDNA, then use the cDNA as a template, and use the following sequence as a primer to obtain the gene sequence of the β-thymosin disease resistance gene by PCR. The sequence of the primer is:

[0026] TroTβ-F: 5'-gatatcATGAGTGACAACAAGCCC-3';

[0027] TroTβ-R: 5'-gatatcTGATGACTCTGACTTCTCC-3'.

[0028] Reaction conditions: pre-denaturation at 94°C for 2 minutes, 30 cycles at 94°C for 30s, 52°C for 30s, and 72°C for 30s, a total of 35 cycles, and finally extension at 72°C for 5 minutes. The amplified fragments were recovered by gel, ligated with pMD19-T vector, transformed into Escherichia coli DH5α, and positive colonies were picked for PCR detection. After the detection is correct, the recombinant p...

Embodiment 2

[0033] The bacteriostasis of embodiment 2β-thymosin

[0034]Dilute the purified β-thymosin protein described in Example 1 to 1 ug / ul in PBS. Edwardsiella tarda, Vibrio harveii and Streptococcus agalactiae were cultured in LB medium at 30°C with shaking at 200rpm / min until OD 600 About 0.6, respectively take 50ul bacterial liquid and 50ul β-thymosin diluent to mix, at 30°C, 250rpm speed, Bioscreen C of Growth Curves Company to measure, OD600 is measured once per hour, continuous measurement for 96 hours, β-thymus The protein can significantly inhibit the growth of Edwardsiella tardiformis, Vibrio harveii and Streptococcus agalactiae, the results are shown in figure 2 .

Embodiment 3

[0035] After embodiment 3 β-thymosin protein injects fish, the resistance to disease and infection of fish is significantly improved

[0036] Dilute the β-thymosin protein purified in Example 1 to 200ug / ml in PBS, which is the β-thymosin dilution. Thirty oval pomfrets (weighing about 15g) were randomly divided into 2 groups, 15 in each group. The two groups were named A and B respectively, each fish in group A was injected with 100ul beta-thymosin diluent, and group B was injected with 100ul PBS.

[0037] Cultivate Edwardsiella tarda in LB medium at 30°C with shaking at 200rpm / min to OD 600 About 0.6, estimated 1OD=5*10 8 CFU / ml, diluted to 10 in PBS 6 CFU / ml is the bacterial suspension of Edwardsiella tardigrade.

[0038] Attack infection

[0039] After injecting 100ul of β-thymosin dilution or 100ul of PBS to the experimental fish of groups A and B for 1 day, each fish was injected with 100ul of the bacterial suspension of Edwardsiella brachii prepared in the above step...

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Abstract

The invention provides trachinotus ovatus beta-thymosin and application thereof, and relates to the field of molecular biology. The cDNA nucleotide sequence of the trachinotus ovatus beta-thymosin is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2. The invention also provides application of the trachinotus ovatus beta-thymosin to anti-bacterial disease medicine, and a recombinant protein expression method of the trachinotus ovatus beta-thymosin. The beta-thymosin provided by the invention can obviously inhibit the growth of pathogenic bacteria; after the injection into the fish is finished, the fish disease-resistant capability can be obviously improved.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to an oval pomfret β-thymosin and its application as an immune enhancer in antibacterial disease performance. Background technique [0002] The pomfret (Trachinotus ovatus) is an important mariculture fish in southern coastal areas such as Hainan, Guangdong and Guangxi in my country. With the continuous expansion of aquaculture scale, the occurrence of diseases is becoming more and more serious. Thymus is one of the important central immune organs in the body's immune system. Thymosin is a polypeptide substance with living activity that was first isolated from fetal bovine thymus protein extract by Goldstein and White in 1966. Among them, the structure of β-thymosin is highly conserved, consisting of 40-44 amino acid residues, and the molecular mass is about 5.0kD. Thymosin not only has immunoregulatory activity and promotes the maturation of T cells, but also plays an important ro...

Claims

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Application Information

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IPC IPC(8): C07K14/575C12N15/16C12N15/70A61K38/32A61P31/04
CPCA61K38/00C07K14/57581C12N15/70
Inventor 孙云周永灿陈晓娟
Owner HAINAN UNIVERSITY
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