Gene, protein, plasmid and application of grouper apoptosis regulatory factor Bok

Abstract

The invention relates to a gene, a protein and a plasmid of a humpback perch apoptosis regulating factor Bok and application of the gene, the protein and the plasmid, and belongs to the field of molecular biology, the sequence of cDNA nucleotide of the humpback perch apoptosis regulating factor Bok is as shown in SEQ ID NO.1, and the amino acid sequence is as shown in SEQ ID NO.2. The invention further provides a construction method of the eukaryotic expression plasmid of the humpback perch apoptosis regulating factor Bok and application of the eukaryotic expression plasmid of the humpback perch apoptosis regulating factor Bok. The regulation effect of the constructed eukaryotic expression vector on other apoptosis-related factors of the humpback perch is verified, and after the eukaryotic expression plasmid of the apoptosis regulatory factor Bok of the humpback perch is injected into a fish body, the disease resistance of the fish body can be remarkably improved, and the eukaryotic expression plasmid has a remarkable immune protection effect.

Description

Technical Field

[0001] The invention relates to the field of molecular biology, and specifically relates to a humpback seabass apoptosis regulating factor Bok (CaBok) and application of the humpback seabass as an immunopotentiator in resisting bacterial diseases. Background Art

[0002] Humpback perch ( Chromileptes altivelis ) is a precious grouper species, known as top seafood, with high economic value. With the breakthrough in the artificial breeding technology of humpback perch, the humpback perch farming industry has broad prospects, but with the increase in breeding density and the deterioration of the breeding environment, diseases frequently occur during the breeding process of humpback perch, causing serious economic losses. At present, the disease is mainly prevented and controlled by the use of drugs, but the excessive use of drugs will cause secondary pollution to the breeding environment and affect the safety of aquatic products. Therefore, starting from the autoimmunity of humpback perch, improving its autoimmunity and then improving its ability to resist pathogen infection is of great significance to the sustainable and green development of my country's aquaculture industry.

[0003] Apoptosis, as a spontaneous programmed cell death process, plays an important role in biological development, maintaining the homeostasis of the internal environment, and regulating immune responses. As a key regulator of the apoptosis process, Bcl2 family proteins have been a hot topic of research since their discovery, focusing mainly on the functions and regulatory mechanisms of Bcl2 family proteins in the apoptosis process. Existing studies have shown that Bcl2 family proteins mainly regulate apoptosis through two endogenous apoptosis regulatory pathways: the mitochondrial regulatory pathway and the endoplasmic reticulum regulatory pathway. They play an important role in regulating the release of apoptotic factors, regulating DNA integrity, and Ca. 2+ It regulates cell apoptosis in many aspects, such as the regulation of protein levels.

[0004] According to the structure and function of different Bcl2 family proteins, Bcl2 family proteins can be divided into three categories: anti-apoptotic proteins containing four BH domains (BH1-4), pro-apoptotic proteins containing four BH domains (BH 1-4), and pro-apoptotic proteins containing only BH 3 domains. Bok contains four BH functional domains. As a pro-apoptotic protein in the Bcl2 family, it plays an important role in the regulation of cell apoptosis. In addition to regulating cell apoptosis, Bok can also participate in the anti-disease immune regulation response of organisms. Studies have shown that Bok plays an important role in the anti-disease immune response of mammals and can be used as an emerging target for cancer treatment, with broad application prospects. However, the function and application potential of Bok in fish still need further study. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a gene, protein, vector of humpback seabass apoptosis regulating factor Bok (CaBok) and its application in humpback seabass anti-bacterial disease drugs, so as to find new strategies for the prevention and control of aquatic animal diseases and promote the sustainable and green development of my country's aquaculture industry.

[0006] The present invention is achieved through the following technical solutions: A humpback bass apoptosis regulator Bok (CaBok) gene, whose cDNA nucleotide sequence is shown in SEQ ID NO.1: ATGGAGATGTTGCGTCGCTCTTCTGTGTTTGCGGCTGAAGTGTTTGACCGCTCGCCCACCGACAAGGAGCTGGTGTCCCAGGCCAAAGCACTGTGCAGAGACTACATCCACTCCAGGCTGAACCGTGCCGGGATAGGCTGGTCTAAACCTGAGCATGGACTGGCTGCATCAGGTGGGGCTCTGGGAGAGATATCATCGGTGCTGCTGTGGCTGGGTGACGAGCTGGAG TACCTTCGACCCAACGTTTTACCGCAACGTAGCGCGACAGCTCAACATCACCGTGGCGTCAGAGAGCATTGTGTCTGATGCTTTCCTGGCTGTGGCTGCAGACATTTTCTCTACAGGTGTGACGTGGGGGAAGGTGGTGTCTTTGTACGCCGTGGCTGGAGCCTTGGCAGTGGACTGCGTTCGCCACGGTCATCCAGCTATGG TCCATACCATTGTTGATTGCATGGGGGAGTTTGTCCGCAAAAGTCTGACCTCCTGGTTGAAGAGGAGAGGTGGCTGGGTGGATGTAACAAAGTGCGTGGTGAACACTGATCCCAGCTTCCGCTCTCACTGGCTGGTGTGTGCCGTCTGTGCCTTTGGACACTACGTGAAGGCCATTGTGTTATACCTCTTCAGGGAGAAGTGA The gene encodes the humpback seabass apoptosis regulator Bok (CaBok) protein, the amino acid sequence of which is shown in SEQ ID NO.2: MEMLRRSSVFAAEVFDRSPTDKELVSQAKALCRDYIHSRLNRAGIGWSKPEHGLAASGGALGEISSVLLWLGDELEYLRPNVYRNVARQLNITVASESIVSDAFL AVAADIFSTGVTWGKVVSLYAVAGALAVDCVRHGHPAMVHTIVDCMGEFVRKSLTSWLKRRGGWVDVTKCVVNTDPSFRSHWLVCAVCAFGHYVKAIVLYLFREK A recombinant vector comprising the gene of the humpback seabass apoptosis regulating factor Bok.

[0007] A recombinant plasmid comprises the recombinant vector.

[0008] The present invention also provides application of the humpback seabass apoptosis regulating factor Bok (CaBok) gene in preparing a humpback seabass anti-bacterial infection preparation.

[0009] Further, the bacterium is Vibrio harveyi ( Vibrio harveyi ).

[0010] The cloning method of the humpback seabass apoptosis regulator Bok (CaBok) gene of the present invention is: extracting total RNA from the head and kidney tissues of the humpback seabass, and reversely transcribing the RNA to obtain cDNA, and then using the cDNA as a template, PCR amplification is performed through the following primer sequences to obtain a full-length sequence, which is 633 bp in length. The sequences of the primers are: CaBok-F:5'- cccggggccacc ATGGAGATGTTGCGTCGCT-3' CaBok-R:5'- cccggg TCACTTCTCCCTGAAGAGGTATAAC-3' PCR reaction program: 95°C pre-denaturation for 5 min, 95°C for 30 s, 58°C for 30 s, 72°C for 1 min, for a total of 32 cycles, and finally extension at 72°C for 5 min.

[0011] The eukaryotic expression plasmid pCaBok of the humpback seabass apoptosis regulator Bok (CaBok) gene is based on the humpback seabass cDNA as a template, and is amplified by PCR using primers CaBok-F and CaBok-R. The amplified fragment is purified and connected to the pEASYS-T1 Simple vector, and transformed into Escherichia coli DH5α competent cells. After plate culture, colonies are picked for PCR detection; the positive colonies detected correctly are activated and the recombinant plasmid is extracted. At the same time, the eukaryotic expression vector pCN3 plasmid is extracted, and the use EcoRThe two plasmids were digested with V restriction endonuclease and recovered; the two plasmids were connected using T4 DNA ligase to construct a recombinant eukaryotic expression plasmid; the recombinant eukaryotic expression plasmid was verified to contain the Bok gene by gene sequencing and was named pCaBok.

[0012] The beneficial effects of the present invention compared with the prior art are as follows: The humpback seabass apoptosis regulating factor Bok (CaBok) eukaryotic expression plasmid of the present invention can promote the expression of other apoptosis regulating factors after being injected into the fish body, and can significantly improve the survival rate of the fish body after Vibrio harveyi infection, and has a significant immune protection effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a diagram for detecting the immune protection effect of the CaBok eukaryotic expression plasmid pCaBok; Figure 2 This is an experimental diagram showing the effect of CaBok eukaryotic expression plasmid pCaBok on the expression changes of other apoptotic factors in the liver tissue of humpback sea bass; Figure 3 This is an experimental diagram showing the effect of CaBok eukaryotic expression plasmid pCaBok on the expression changes of other apoptotic factors in the spleen tissue of humpback seabass; Figure 4 This is an experimental diagram showing the effect of the CaBok eukaryotic expression plasmid pCaBok on the expression changes of other apoptotic factors in the head and kidney tissues of humpback seabass. DETAILED DESCRIPTION

[0014] The technical solution of the present invention is further explained below by examples, but the technical solution of the present invention is not limited in any form by the examples.

[0015] Example 1: Cloning of the gene for the humpback seabass apoptosis regulator Bok (CaBok): (1) Total RNA was extracted from the head and kidney tissues of humpback seabass using the Promega total RNA extraction kit.

[0016] (2) Using the total RNA extracted above as a template, use the Promega reverse transcription kit (GoScript™ Reverse Transcription Mix, Oligo(dT), A2791) to synthesize the first-strand cDNA. The steps are as follows: 1) Prepare the mixed solution as shown in Table 1: Table 1 Mixed liquid configuration system 2) After mixing, place in PCR instrument and set the reverse transcription program as shown in Table 2: Table 2 PCR reverse transcription program 3) After the procedure is completed, the obtained cDNA is stored at -20 ℃ for future use.

[0017] (3) Cloning of the gene encoding the apoptosis regulator Bok (CaBok) in humpback seabass The ORF sequence of apoptosis regulator Bok was obtained from the humpback seabass transcriptome library, and the following primers were designed using the primer design software Primer5.0 for cloning the CaBok gene: CaBok-F:5'- cccggggccacc ATGGAGATGTTGCGTCGCT-3' CaBok-R:5'- cccggg TCACTTCTCCCTGAAGAGGTATAAC-3' PCR reaction system and procedure: 1) Configure the mixed system as shown in Table 3: Table 3 PCR mixture configuration system 2) After mixing, place in PCR instrument and set PCR amplification program: first pre-denaturation at 95 ℃ for 5 min; then denaturation at 95 ℃ for 30 s, annealing at 58 ℃ for 30 s, extension at 72 ℃ for 1 min, for a total of 32 cycles; finally, extension at 72 ℃ for 5 min.

[0018] 3) After the PCR reaction, the amplified product was detected using 1.2% agarose gel, and then the PCR product was purified using the FastPure ® Gel DNA Extraction Mini Kit of Nanjing Novogene Biotech Co., Ltd.

[0019] (4)TA cloning: 1) Carrier connection pEASY ® -T1 simple Cloning Vector Kit was used to connect the above-mentioned purified product with the T1 vector. The connection system is shown in Table 4. After the system is mixed, it is incubated at room temperature for 10 min.

[0020] Table 4 Connection system 2) Conversion Take out the DH5α competent cells from -80 ℃ and place them on ice to thaw, then add 6 μL of the above ligation product, mix gently and place on ice for 30 min.

[0021] Then transfer to a 42 °C water bath for heat shock for 90 s and continue to ice bath for 5 min after the heat shock.

[0022] After the ice bath, add 800 μL LB liquid medium and place in an incubator for shaking culture at 37 °C and 180 rpm for 1 h.

[0023] At the end of the culture, centrifuge at 4000 × g for 5 min to collect the bacteria, then discard 750 μL of the supernatant, use the remaining supernatant to resuspend the bacteria and spread them on solid LB medium containing Amp antibiotics and IPTG / X-GalLB, and place it in a 37 °C incubator for inverted culture for 12 h.

[0024] 3) Positive clone screening and detection Add 10 μL sterile ddH2O to a 200 μL PCR tube, pick a monoclonal colony (white spot) into the PCR tube, and mix by pipetting. Use the mixed bacterial solution as a template and primers CaBok-F / R for PCR detection. The PCR detection system is the same as above. After the PCR reaction is completed, use 1% agarose gel electrophoresis to detect the PCR product, culture the positive strains and send them for sequencing, and store the strains with correct sequencing at -80 ℃.

[0025] Example 2: Construction of eukaryotic expression vector of humpback seabass apoptosis regulator Bok (CaBok).

[0026] (1) Plasmid extraction: The correctly sequenced CaBok plasmid strain and the eukaryotic expression vector pCN3 plasmid strain were revived and cultured, and then the two plasmids were extracted using the Plasmid Mini Kit I plasmid extraction kit from Omega.

[0027] (2) Enzyme digestion: use EcoR The two plasmids extracted above were digested with restriction endonucleases, and the digestion system is shown in Table 5: Table 5 Enzyme digestion system The mixed system was placed in a 37°C constant temperature metal bath and incubated for 30 min. After the CaBok plasmid incubation is completed, agarose electrophoresis can be directly performed to detect the enzyme digestion results. After the pCN3 vector is incubated for 20 min, 3 μL FastAP needs to be added to the enzyme digestion system and incubated for another 10 min before the enzyme digestion results are detected.

[0028] (3) Use the FastPure ® Gel DNA Extraction Mini Kit from Nanjing Novezan Biotechnology Co., Ltd. to purify the enzyme digestion product.

[0029] (4) Vector ligation and transformation 1) Use T4 DNA ligase to ligate the digested CaBok and pCN3. The ligation system is shown in Table 6. Mix well and incubate in a 16 ℃ constant temperature metal bath overnight.

[0030] Table 6 Connection system 2) Conversion Take out the DH5α competent cells from -80 ℃ and place them on ice to thaw, then add 6 μL of the above ligation product, mix gently and place on ice for 30 min.

[0031] Then transfer to a 42 °C water bath for heat shock for 90 s and continue to ice bath for 5 min after the heat shock.

[0032] After the ice bath, add 800 μL LB liquid medium and place in an incubator for shaking culture at 37 °C and 180 rpm for 1 h.

[0033] At the end of the culture, centrifuge at 4000 × g for 5 min to collect the bacteria, then discard 750 μL of the supernatant, use the remaining supernatant to resuspend the bacteria and spread them on solid LB medium containing Amp antibiotics and IPTG / X-GalLB, and place it in a 37 °C incubator for inverted culture for 12 h.

[0034] 3) Positive clone screening and detection Add 10 μL sterile ddH2O to a 200 μL PCR tube, pick up a monoclonal colony (white spot) into the PCR tube, and mix by pipetting. Use the mixed bacterial solution as a template and primers CaBok-F / R for PCR detection. The PCR detection system is the same as above. After the PCR reaction is completed, use 1% agarose gel electrophoresis to detect the PCR product, culture the positive strains and send them for sequencing, and store the strains with correct sequencing at -80 ℃.

[0035] The primers used were: CaBok-F:5'-cccggggccaccATGGAGATGTTGCGTCGCT-3' CaBok-R:5'-cccgggTCACTTCTCCCTGAAGAGGTATAAC -3' CN-F:5'-CTTGCGTTTCTGATAGGCACCTA-3' CN-R:5'-TGCGGGCCTCTTCGCTATT-3' Example 3: Application of humpback bass apoptosis regulator Bok (CaBok) eukaryotic expression plasmid pCaBok (1) The pCaBok plasmid strain with the correct sequencing results and the pCN3 plasmid strain were cultured in large quantities, and the endotoxin-free plasmids of pCN3 and pCaBok were extracted using the endotoxin-free plasmid extraction kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. and stored at -20°C after extraction.

[0036] (2) The pCaBok and pCN3 endotoxin-free plasmids extracted above were diluted to 150 μg / mL with PBS. Then 60 humpback sea bass were randomly divided into three groups, with 20 in each group. The three groups were injected intramuscularly with 100 μL of the diluted pCaBok, pCN3 and PBS (control group) respectively.

[0037] (3) Inoculate Vibrio harveyi into LB medium and culture at 30°C with shaking at 200 rpm / min until OD 600 The final concentration of the bacterial solution was about 0.8, and PBS was used to dilute the bacterial solution to 10 6 CFU / ml, that is, Vibrio harveyi bacterial suspension.

[0038] (4) 15 days after plasmid injection, the three groups of fish were intraperitoneally injected with 100 μL of the above-mentioned Vibrio harveyi bacterial suspension for simulated infection. Subsequently, the number of deaths of humpback seabass in each group was continuously observed and recorded for 10 days after infection. After 10 days, the total number of deaths of fish in each group was counted: pCaBok group, 6; pCN3 group, 10; PBS group, 11. The survival rates of pCaBok group, pCN3 group, and PBS group were 70%, 50%, and 45%, respectively. The survival rate of fish in the pCaBok group was significantly higher than that in the control group and the pCN3 group.

[0039] These results indicate that pCaBok can significantly enhance the ability of humpback seabass to resist infection by Vibrio harveyi and has a good immune protection effect.

[0040] Example 4: Effect of the humpback bass apoptosis regulator Bok (CaBok) eukaryotic expression plasmid pCaBok on the expression of other apoptosis factors (1) The pCaBok plasmid strain with the correct sequencing results and the pCN3 plasmid strain were cultured in large quantities, and the endotoxin-free plasmids of pCN3 and pCaBok were extracted using the endotoxin-free plasmid extraction kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. and stored at -20°C after extraction.

[0041] (2) The pCaBok and pCN3 endotoxin-free plasmids extracted above were diluted to 150 μg / mL with PBS. Then, 15 humpback sea bass were randomly divided into three groups, with 5 in each group. The three groups were injected intramuscularly with 100 μL of the diluted pCaBok, pCN3, and PBS (control group) respectively.

[0042] (3) On the 15th day after plasmid injection, the liver, spleen and head kidney tissues of the three groups of fish were collected to extract RNA and reversely converted into cDNA for qRT-PCR (for specific methods, see Zhengshi Zhang, Xiucong Hu, Qianying Diao, Panpan Zhang, Ying Wu, Zhenjie Cao, Yongcan Zhou, Chunsheng Liu, Yun Sun. Macrophage migration inhibitory factor (MIF) of golden pompano ( Trachinotus ovatus ) is involved in the antibacterial immune response. Developmental and Comparative Immunology, 2022, 133: 104445), and the expression of other apoptotic factors was detected. The results showed that the expression levels of Cyt c, Caspase3 and Caspase9 in the liver, spleen and head kidney tissues of the pCaBok group were significantly higher than those in the control group ( P <0.05); however, there were no significant differences in the expression of other apoptotic factors in the liver, spleen, and head kidney tissues of the pCN3 group compared with the control group ( P >0.05).

[0043] These results indicate that pCaBok regulates the expression of other apoptotic factors in humpback seabass.

Claims

1. A humpback seabass apoptosis regulatory factor Bok gene, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The gene encoding the humpback seabass apoptosis regulator Bok protein according to claim 1, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. A recombinant vector, characterized in that: The recombinant vector contains the gene of the humpback seabass apoptosis regulating factor Bok as described in claim 1.

4. A recombinant plasmid, characterized in that: The recombinant plasmid comprises the recombinant vector according to claim 3.

5. An application of the Bok gene, a humpback seabass apoptosis regulator, characterized in that: The application is to prepare a humpback bass anti-bacterial infection preparation using the humpback bass apoptosis regulatory factor Bok gene, and the bacteria is Vibrio harveyi.

6. The use of the recombinant plasmid according to claim 4, characterized in that: The application is to prepare a humpback seabass anti-bacterial infection preparation using a recombinant plasmid, wherein the bacterium is Vibrio harveyi.

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