A kind of oval pompano β-thymosin and its application
A technology of oval pomfret and thymosin, applied in the field of molecular biology, can solve the problems of unclear function and application potential of β-thymosin, and achieve the effect of improving disease resistance
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[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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