Gene, protein, plasmid and application of trachinotus ovatus apoptosis regulatory factor Bok

Abstract

The invention relates to a gene, a protein and a plasmid of a trachinotus ovatus apoptosis regulatory 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 trachinotus ovatus apoptosis regulatory 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 trachinotus ovatus apoptosis regulatory factor Bok. The regulation effect of the constructed eukaryotic expression vector on other apoptosis-related factors of the trachinotus ovatus is verified, and after the eukaryotic expression plasmid for the apoptosis regulation factor Bok of the trachinotus ovatus is injected into a fish body, the disease resistance of the fish body can be remarkably improved, and a remarkable immune protection effect is achieved.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to a pomfret apoptosis regulator Bok (TroBok) and its application as an immune enhancer in pomfret's resistance to bacterial diseases. Background Technology

[0002] Oval pomfret ( Trachinotus ovatus As one of the most valuable marine economic fish species along my country's coast, the oval pomfret possesses significant economic value. However, with increasing farming density and deteriorating aquaculture environments, diseases frequently occur during oval pomfret farming, causing severe economic losses to my country's aquaculture industry. Furthermore, excessive use of medications for prevention and treatment can cause secondary pollution to the farming environment and also impact food safety. Therefore, improving the oval pomfret's own immunity, thereby enhancing its resistance to pathogen infection, is extremely important for the sustainable development of my country's aquaculture industry.

[0003] Apoptosis is a spontaneous programmed cell death process that plays a crucial role in the development of organisms, the regulation of homeostasis, and immune regulation. Bcl2 family proteins are key factors regulating apoptosis, and their functions and regulatory mechanisms have been a hot research topic. Existing studies have shown that Bcl2 family proteins primarily regulate apoptosis through endogenous apoptosis regulatory pathways, including mitochondrial and endoplasmic reticulum pathways, affecting the release of apoptotic factors, DNA integrity, and calcium. 2+ Apoptosis is regulated in multiple ways, including by adjusting the level of cells.

[0004] Based on the structure and function of different members of the Bcl2 family of proteins, they can be divided into three categories: anti-apoptotic proteins containing four BH domains (BH 1-4), pro-apoptotic proteins containing four BH domains (BH 1-4), and pro-apoptotic proteins containing only the BH 3 domain. Bok is a pro-apoptotic protein in the Bcl2 family that plays an important role in regulating apoptosis. In addition to regulating apoptosis, Bok can also participate in the regulation of disease-fighting immune responses in organisms. Studies have shown that Bok plays an important role in the disease-fighting immune response in mammals and may serve as a promising new target for cancer treatment. However, the function and application potential of Bok in fish still require further investigation. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide the gene, protein, and vector of the apoptosis regulator Bok (TroBok) in pomfret and its application in antibacterial disease drugs in pomfret, so as to find new strategies for the prevention and control of aquatic animal diseases and promote the healthy and sustainable development of my country's aquaculture industry.

[0006] This invention is achieved through the following technical solution: A gene for regulating apoptosis in the oval pomfret, Bok (TroBok), has a cDNA nucleotide sequence shown in SEQ ID NO. 1: . The gene encodes the Bok (TroBok) protein, a regulator of apoptosis in the oval pomfret, and the amino acid sequence of the protein is shown in SEQ ID NO.2: MEVLRRSSVFAAEVLDVFDRSLTEKELVSQSKALCRDYILSRLNQNGLGWSKTELNLSPSNAALAEVSLVLLCLGDELECIQPSLYRNVARQLNISVAMENMVSDAF IGVATEIFSTGITWGKVVAMYAVAGALAVDCVRQGHPTSVHILVDSLGQFVRKFLVPWLKRRGGWAEITKCVVKRDLTPEHHWLSSVIESLKYFLTTMYVYIMKEP. A recombinant vector containing the gene of Bok, the apoptosis regulator of the oval pomfret.

[0007] A recombinant plasmid containing the recombinant vector.

[0008] The present invention also provides the application of the apoptosis regulator Bok (TroBok) gene of the oval pomfret in the preparation of an antibacterial agent of the oval pomfret.

[0009] Furthermore, the bacteria is Vibrio harveyi ( Vibrio harveyi ).

[0010] The cloning method for the apoptosis regulator Bok (TroBok) gene of the oval pomfret described in this invention is as follows: Total RNA is extracted from the head kidney tissue of the oval pomfret, and cDNA is obtained by reverse transcription of the RNA. Then, using the cDNA as a template, PCR amplification is performed using the primer sequences described below to obtain the full-length sequence, which is 642 bp in length. The primer sequences are as follows: TroBok-F:5'- gatatcgccacc ATGGAGGTCCTGCGGAGGTCTTCT-3' TroBok-R:5'- gatatc TGGCTCCTTCATGATGTAGACG-3' PCR reaction program: 95℃ pre-denaturation for 5 min, 95℃ for 30 s, 58℃ for 30 s, 72℃ for 1 min, for a total of 32 cycles, and finally extension at 72℃ for 5 min.

[0011] The eukaryotic expression plasmid pTroBok, representing the apoptosis regulator factor Bok (TroBok) gene from the oval pomfret, was amplified by PCR using oval pomfret cDNA as a template and primers TroBok-F and TroBok-R. The amplified fragment was purified and ligated into the pEASYS-T1 Simple vector, then transformed into *E. coli* DH5α competent cells. Positive colonies were selected for PCR detection after plating. The correctly detected positive colonies were activated, and the recombinant plasmid was extracted. Simultaneously, the eukaryotic expression vector pCN3 was extracted and used separately. EcoR The two plasmids were digested and recovered using V restriction endonuclease; the two plasmids were ligated using T4 DNA ligase to construct a recombinant eukaryotic expression plasmid; gene sequencing confirmed that the recombinant eukaryotic expression plasmid contained the Bok gene, and it was named pTroBok.

[0012] The beneficial effects of this invention compared to the prior art are as follows: The eukaryotic expression plasmid of the oval pomfret apoptosis regulator Bok (TroBok) of this invention, when injected into fish, can promote the expression of other pro-apoptotic regulators, reduce the expression of anti-apoptotic factors, and significantly improve the survival rate of fish infected with Vibrio harveyi, thus exhibiting a significant immunoprotective effect. Attached Figure Description

[0013] Figure 1 A graph showing the immunoprotective effect of the TroBok eukaryotic expression plasmid pTroBok. Figure 2 Figure 1 shows the effect of the TroBok eukaryotic expression plasmid pTroBok on the expression of other apoptosis factors in the liver tissue of the oval pomfret. Figure 3 Figure 1 shows the effect of the TroBok eukaryotic expression plasmid pTroBok on the expression of other apoptosis factors in the spleen tissue of oval pomfret. Figure 4 This figure shows the effect of the TroBok eukaryotic expression plasmid pTroBok on the expression of other apoptosis factors in the head kidney tissue of the oval pomfret. Detailed Implementation

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

[0015] Example 1: Cloning of the TroBok gene, apoptosis regulator in oval pomfret: (1) Total RNA was extracted using the Promega Total RNA Extraction Kit.

[0016] (2) Using the total RNA extracted above as a template, the first strand of cDNA was synthesized using the Promega reverse transcription kit (GoScript™ Reverse Transcription Mix, Oligo(dT), A2791). The procedure is as follows: 1) Prepare the mixture as shown in Table 1: Table 1 Mixture Preparation System 2) After mixing, place the mixture into a 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, store the obtained cDNA at -20 ℃ for later use.

[0017] (3) Cloning of the TroBok gene, apoptosis regulator in oval pomfret. The ORF sequence of the apoptosis regulator Bok was retrieved from the oval pomfret transcriptome library. Primers were designed using Primer 5.0 software as follows for cloning the TroBok gene: TroBok-F:5'- gatatcgccacc ATGGAGGTCCTGCGGAGGTCTTCT-3' TroBok-R:5'- gatatc TGGCTCCTTCATGATGTAGACG-3' PCR reaction system and procedure: 1) Configure the mixing system as shown in Table 3: Table 3 PCR Mixture Preparation System 2) After mixing, place the mixture into a PCR instrument and set the PCR amplification program as follows: pre-denaturation 95 ℃ for 5 min; denaturation 95 ℃ for 30 s, annealing 58 ℃ for 30 s, extension 72 ℃ for 1 min, 32 cycles; extension 72 ℃ for 5 min; store at 4 ℃.

[0018] 3) After the PCR reaction was completed, the amplification products were detected using a 1.2% agarose gel, and then the products were purified using the FastPure® Gel DNA Extraction Mini Kit from Nanjing Novizan Biotechnology Co., Ltd.

[0019] (4) TA cloning: 1) Carrier connection Using pEASY from Beijing Quanshijin Biotechnology Co., Ltd. ®The T1 simple Cloning Vector kit was used to ligate the purified product to the T1 vector. The ligation system is shown in Table 4. After mixing, the system was incubated at room temperature for 10 min. Table 4 Connection System 2) Transformation DH5α competent cells were removed from -80 °C and thawed on ice. Then, 6 μL of the above ligation product was added, gently mixed, and incubated on ice for 30 min.

[0020] Then, it was transferred to a 42 ℃ water bath for heat shock for 90 s, and after the heat shock was completed, it was continued in an ice bath for 5 min.

[0021] After the ice bath, add 800 μL of LB liquid medium and incubate in an incubator at 37 °C and 180 rpm for 1 h with shaking.

[0022] After the culture was completed, the bacterial cells were collected by centrifugation at 4000 ×g for 5 min. Then, 750 μL of supernatant was discarded, and the bacterial cells were resuspended in the remaining supernatant and spread onto solid LB medium containing Amp antibiotic and IPTG / X-GalLB. The medium was then incubated upside down in a 37 ℃ incubator for 12 h.

[0023] 3) Screening and detection of positive clones Add 10 μL of sterile ddH2O to a 200 μL PCR tube, pick a single colony (white spot) and mix well by pipetting. Using the mixed bacterial solution as a template, perform PCR detection using primers TroBok-F / R, following the same PCR detection system as above. After the PCR reaction, detect the PCR products using 1% agarose gel electrophoresis. Culture and preserve positive strains and send them for sequencing. Store correctly sequenced strains at -80 ℃.

[0024] Example 2: Construction of a eukaryotic expression vector for Bok (TroBok), an apoptosis regulator in oval pomfret.

[0025] (1) Plasmid extraction: The TroBok plasmid strain and the pCN3 eukaryotic expression vector strain with correct sequencing were revived and cultured, and then plasmids were extracted using the Plasmid Mini Kit I plasmid extraction kit from Omega.

[0026] (2) Enzyme digestion: The two plasmids extracted above were used EcoR Restriction endonucleases (V) were used for digestion, and the digestion system is shown in Table 5. Table 5 Enzyme digestion system The mixed system was incubated in a 37 ℃ constant temperature metal bath for 30 min. After the TroBok plasmid was incubated, agarose gel electrophoresis was performed directly to detect the enzyme digestion results. After the pCN3 vector was incubated for 20 min, 3 μL FastAP was added to the enzyme digestion system and incubated for another 10 min before the enzyme digestion results were detected.

[0027] (3) The enzyme digestion products were purified using the FastPure® Gel DNA Extraction Mini Kit from Nanjing Novizan Biotechnology Co., Ltd.

[0028] (4) Carrier connection and conversion 1) Use T4 DNA ligase to ligate the digested TroBok and pCN3. The ligation system is shown in Table 6. After mixing, incubate overnight in a 16 ℃ constant temperature metal bath.

[0029] Table 6 Connection System 2) Transformation DH5α competent cells were removed from -80 °C and thawed on ice. Then, 6 μL of the above ligation product was added, gently mixed, and incubated on ice for 30 min.

[0030] Then, it was transferred to a 42 ℃ water bath for heat shock for 90 s, and after the heat shock was completed, it was continued in an ice bath for 5 min.

[0031] After the ice bath, add 800 μL of LB liquid medium and incubate in an incubator at 37 °C and 180 rpm for 1 h with shaking.

[0032] After the culture was completed, the bacterial cells were collected by centrifugation at 4000 ×g for 5 min. Then, 750 μL of supernatant was discarded, and the bacterial cells were resuspended in the remaining supernatant and spread onto solid LB medium containing Amp antibiotic and IPTG / X-GalLB. The medium was then incubated upside down in a 37 ℃ incubator for 12 h.

[0033] 3) Screening and detection of positive clones Add 10 μL of sterile ddH2O to a 200 μL PCR tube, pick a single colony (white spot) and mix well by pipetting. Using the mixed bacterial solution as a template, perform PCR detection using primers TroBok-F / R, following the same PCR detection system as above. After the PCR reaction, detect the PCR products using 1% agarose gel electrophoresis. Culture and preserve positive strains and send them for sequencing. Store correctly sequenced strains at -80 ℃.

[0034] The primers used are: TroBok-F:5'- gatatcgccacc ATGGAGGTCCTGCGGAGGTCTTCT-3' TroBok-R:5'- gatatc TGGCTCCTTCATGATGTAGACG-3' CN-F:5'-CTTGCGTTTCTGATAGGCACCTA-3' CN-R:5'-TGCGGGCCTCTTCGCTATT-3' Example 3: Application of the eukaryotic expression plasmid pTroBok, a regulator of apoptosis in oval pomfret (TroBok). (1) The pTroBok plasmid strain with correct sequencing and the pCN3 plasmid strain were cultured in large quantities, and the endotoxin-free plasmids of pCN3 and pTroBok were extracted using the endotoxin-free plasmid large extraction kit of Tiangen Biotech (Beijing) Co., Ltd. After extraction, they were stored at -20℃.

[0035] (2) The extracted pTroBok and pCN3 endotoxin-free plasmids were diluted to 150 μg / mL with PBS. Subsequently, 60 oval pomfret were randomly divided into three groups of 20 each. The three groups were intramuscularly injected with 100 μL each of the diluted pTroBok, pCN3 and PBS (control group).

[0036] (3) Inoculate Vibrio harveyi into LB medium and culture at 30°C with shaking at 200 rpm / min until OD. 600 The concentration was approximately 0.8, and it was diluted with PBS to a final bacterial concentration of 10. 5 CFU / ml is a suspension of Vibrio harveyi bacteria.

[0037] (4) Fifteen days after plasmid injection, the three groups of fish were intraperitoneally injected with 100 μL of the above-mentioned Vibrio harveyi bacterial suspension to simulate viral challenge. The number of deaths in each group of oval pomfret was then continuously observed and recorded for 10 days after challenge. After 10 days, the total number of deaths in each group was counted: pTroBok group, 7 fish; pCN3 group, 11 fish; PBS group, 10 fish. The survival rates of the pTroBok group, pCN3 group, and PBS group were 65%, 45%, and 50%, respectively. The survival rate of the fish in the pTroBok group was significantly higher than that of the control group and the pCN3 group.

[0038] These results indicate that pTroBok can significantly enhance the resistance of oval pomfret to Vibrio harveyi infection and has a good immunoprotective effect.

[0039] Example 4: Effects of the eukaryotic expression plasmid pTroBok (TroBok), a pampascopula apoptosis regulator, on the expression of other apoptosis factors. (1) The pTroBok plasmid strain with correct sequencing and the pCN3 plasmid strain were cultured in large quantities, and the endotoxin-free plasmids of pCN3 and pTroBok were extracted using the endotoxin-free plasmid large extraction kit of Tiangen Biotech (Beijing) Co., Ltd. After extraction, they were stored at -20℃.

[0040] (2) The extracted pTroBok and pCN3 endotoxin-free plasmids were diluted to 150 μg / mL with PBS. Subsequently, 15 oval pomfret were randomly divided into three groups of 5 fish each. The three groups were intramuscularly injected with 100 μL each of the diluted pTroBok, pCN3 and PBS (control group).

[0041] (3) On day 15 after plasmid injection, liver, spleen, and head kidney tissues from three groups of fish were collected, and RNA was extracted and reversed into cDNA for qRT-PCR (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 (This is involved in the antibacterial immune response. Developmental and Comparative Immunology, 2022, 133: 104445), and the expression of other apoptosis factors was detected. The results showed that the expression levels of pro-apoptotic factors such as p53, Cyt c, Caspase 3, Caspase 7, and Caspase 9 in the liver, spleen, and head kidney tissues of the pTroBok group were significantly higher than those in the control group. P <0.05), while the expression level of the anti-apoptotic factor Bcl2 was significantly lower than that of the control group ( P <0.05); however, the expression levels of other apoptosis factors in the liver, spleen, and head kidney tissues of the pCN3 group were not significantly different from those of the control group. P >0.05).

[0042] These results indicate that pTroBok has a regulatory effect on the expression of other apoptosis factors in pomfret.

Claims

1. A Bok gene, a regulator of apoptosis in the oval pomfret, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The Bok protein, an apoptosis regulator in oval pomfret encoded by the gene of claim 1, is 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 Bok, the apoptosis regulator of oval pomfret as described in claim 1.

4. A recombinant plasmid, characterized in that, The recombinant plasmid contains the recombinant vector of claim 3.

5. The application of the Bok gene, a regulator of apoptosis in the oval pomfret, characterized in that... The application involves preparing an antibacterial agent for pomfret using the Bok gene, an apoptosis regulator of pomfret, wherein the bacteria is Vibrio harveyi.

6. The application of the recombinant plasmid according to claim 4, characterized in that, The application involves using recombinant plasmids to prepare an antibacterial agent for oval pomfret, wherein the bacteria is Vibrio harveyi.

Images