Application of recombinant protein and gene of TNF-α1 and its TNF domain from Tilapia ovata

Abstract

The present invention relates to an application of a recombinant protein and gene of TNF-α1 of oval pomfret and its TNF domain, belonging to the field of molecular biology technology, wherein the gene sequence of TNF-α1 of oval pomfret is shown as SEQ ID NO.1, and the amino acid sequence of the protein encoded by the gene is shown as SEQ ID NO.2, and its motif comprises a TNF domain. The gene sequence of the TNF domain is shown as SEQ ID NO.3, and the amino acid sequence of the protein encoded by it is shown as SEQ ID NO.4. The present invention obtains the TNF domain recombinant protein of TNF-α1 in vitro, and finds that it can significantly promote the apoptosis of liver cancer cells, and after overexpressing or interfering the expression of the TNF domain of TNF-α1, it is found that it can significantly enhance the immune function of oval pomfret against Vibrio harveyi infection, and can be developed into an immune preparation, with good practical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of fish molecular immunology, and particularly relates to the application of ovate pomfret TNF-alpha1 and recombinant protein and gene of TNF domain thereof. Background Art

[0002] In mammals, tumor necrosis factor α (TNF-α) is mainly produced by activated monocytes / macrophages, which can kill and inhibit tumor cells. At the same time, TNF-α is also involved in the regulation of the body's inflammatory response and immune response, and plays an important role in promoting cell growth, differentiation, apoptosis, etc. At present, TNF-α genes have been published in fish such as flounder (Paralichthys olivaceus), rainbow trout (Oncorhynchus mykiss), zebrafish (Danio rerio), large yellow croaker (Larimichthys crocea), carp (Cyprinus carpio), goldfish (Carassius auratus), crucian carp (Carassius auratus), grass carp (Ctenopharyngodonidella) and gilthead sea bream (Sparus aurata). Some of these fish have two types of TNF-α, TNF-α1 / α2. Fish TNF-α can activate immune cells and participate in the body's anti-infection, which is similar to mammals, but there are also differences. For example, goldfish TNF-α1 / α2 can enhance the phagocytosis and respiratory burst response of macrophages and induce the production of nitric oxide by primary goldfish macrophages. Recombinant grass carp TNF-α promotes the gene and protein expression of pIgR through the NF-κB signaling mechanism, thereby enhancing the immune function of grass carp. Recombinant rainbow trout TNF-α can promote the migration and phagocytosis of leukocytes and upregulate the expression of genes such as IL-1, IL-8, TNF-α and COX-2. Recombinant gilthead sea bream TNF-α can induce the rapid aggregation of phagocytic granulocytes to the injection site and the formation of granulocytes in the head kidney tissue, and also trigger the respiratory burst of peritoneal exudate and head kidney leukocytes, but it has no cytotoxicity to mouse L929 fibroblasts. In addition, recombinant gilthead sea bream TNF-α and recombinant carp TNF-α cannot directly activate phagocytes, but indirectly activate phagocytes by stimulating endothelial cells. This shows that TNF-α in fish and other vertebrates is species-specific. Although the TNF-α gene has been identified in many fish, its biological function still needs to be further elucidated.

[0003] Trachinotus ovatus is a farmed fish with important economic value in the coastal areas of southern China and is currently the main species for deep-water cage farming in the south. However, in recent years, various diseases caused by bacteria, viruses and parasites have continued to break out in the farmed population of ovate pomfret, causing huge economic losses. However, current studies have only found that when Cryptocaryon irritans infects ovate pomfret, TNF-α can signal through the NF-κB pathway, and other details are unknown. Therefore, it is necessary to continue to study the methods and means of TNF-α in ovate pomfret to prevent and treat fish diseases and develop useful immune preparations, which have important reference value for the prevention and treatment of fish diseases. Summary of the invention

[0004] In view of the deficiencies of the prior art, the technical problem to be solved by the present invention is to provide a recombinant protein and gene application of tumor necrosis factor α1 (TNF-α1) and its TNF domain of oval pomfret. The present invention constructs a recombinant plasmid and transfers it into an expression strain, uses IPTG to induce the expression of the TNF domain protein of oval pomfret TNF-α1, optimizes the expression conditions, purifies and obtains the target protein, and further verifies that the prepared oval pomfret recombinant protein can promote apoptosis of liver cancer cells and enhance the body's antibacterial function, so it has good practical application value.

[0005] In order to solve the above technical problems, the present invention is implemented by the following technical solutions:

[0006] The present invention provides application of TNF-α1 recombinant protein of silver pomfret, wherein the application is the use of the recombinant protein in preparing a preparation for promoting apoptosis of liver cancer cells Hep G2, and the sequence of the recombinant protein is SEQ ID NO.3.

[0007] The present invention also provides an application of the ovate pomfret TNF-α1 gene, wherein the ovate pomfret TNF-α1 gene is used to prepare an injection preparation containing siTroTNF-α1 against Vibrio ovalis, and the nucleotide sequence of the ovate pomfret TNF-α1 gene is SEQ ID NO.1.

[0008] The present invention further provides an application of a recombinant protein of the TNF domain of TNF-α1 of silver pomfret, wherein the application is the use of the recombinant protein in the preparation of a preparation for promoting apoptosis of liver cancer cells Hep G2, and the sequence of the recombinant protein is SEQ ID NO.4.

[0009] The present invention further provides an application of the TNF domain gene of Vibrio ovalis TNF-α1, wherein the application is to use the TNF domain gene to prepare an injection preparation containing siTroTNF-α1 against Vibrio ovalis, and the nucleotide sequence of the TNF domain gene is SEQ ID NO.3.

[0010] The cDNA nucleotide sequence of the TNF-α1 gene of the oval pomfret is as shown in SEQ ID NO.1:

[0011] .

[0012] The amino acid sequence of the TNF-α1 of the oval pomfret is shown in SEQ ID NO.2:

[0013] MVDYTAAPGDVEMGLEDRSVILVEKKSSTEWIWKVFGTLLIVALCLGAVLLLCGWYRNAPEMTTQSGQIEALMEKDSAEKTDPHYTLRQISSNSKAAIHLEGSYDEDNESSKKELEWKNGQGQAFAQG LKLVNNQIEVPETGLYFVYSQASFRVSCNDEEDKEAGERFTPLSHRIWRYSDSIGGKASLMSAVRSVCQTTAQEEDSYRDGRSWYNTIYLGAVFQLNKGDTLWTETNQLSELDTDEGKTFFGVFAL.

[0014] The cDNA nucleotide sequence of the TNF domain of the ovate pomfret TNF-α1 is shown in SEQ ID NO.3:

[0015] gcagccatccatttagaaggtagctacgatgaagacaatgagagctcgaaaaaggagttggagtggaagaacggtcaaggccaggcattcgctcagggcttgaagttggtgaacaaccag atcgaagtccctgaaaccggcctctactttgtctacagccaggcgtcgttcagagtctcctgcaacgatgaagaggacaaggaggcgggagaacgcttcacacctctcagccacaggatc tggcggtactcagactccataggcggcaaagcatctctgatgagtgcggtgaggtcggtgtgccaaaccactgcccaggaggaggacagttacagagacggacggagctggtacaacacc atttatctgggtgcagtgtttcagctgaataaaggagacacactgtggacggaaaccaaccagctgtcagagctggacaccgatgagggcaagactttctttggtgtgtttgcactttga.

[0016] The amino acid sequence of the TNF domain of the ovate pomfret TNF-α1 is shown in SEQ ID NO.4:

[0017] AAIHLEGSYDEDNESSKKELEWKNGQGQAFAQGLKLVNNQIEVPETGLYFVYSQASFRVSCNDEEDKEAGERFTPLSHRIWRYSDSIGGKASLMSAVRSVCQTTAQEEDSYRDGRSWYNTIYLGAVFQLNKGDTLWTETNQLSELDTDEGKTFFGVFAL.

[0018] The present invention provides a TNF domain gene of TNF-α1 of silver pomfret and a recombinant expression method of the protein encoded by the gene, which specifically comprises extracting RNA from the head and kidney tissue of silver pomfret, reversely transcribing the RNA into cDNA, and then using the cDNA as a template and the following sequence as a primer to obtain the nucleotide sequence of the TNF-α1 domain gene by PCR, wherein the sequence of the primer is:

[0019] TroTNF-α1-F: 5'-gatatcgccaccATGGCAGCCATCCATTTAGAAGGTAG-3';

[0020] TroTNF-α1-R: 5'-gatatcAAGTGCAAACACACCAAAGAAAGT-3'.

[0021] The nucleotide sequence of the TNF-α1 domain gene obtained by PCR was connected to the pMD19-T vector and transformed into Escherichia coli DH5α. The recombinant was sequenced and identified. After the sequencing was correct, it was named TroTNF-α1-Tsimp le.

[0022] The plasmid is extracted, and a 483 bp fragment is recovered after digestion with EcoR V restriction endonuclease; pET-32a plasmid is extracted, and after digestion with EcoR V, the 483 bp recovered fragment is connected to pET-32a using T4 ligase to construct a recombinant plasmid; the recombinant plasmid is verified to contain the TNF-α1 domain by gene sequencing, and is named pET32a-TroTNF-α1; the recombinant pET32a-TroTNF-α1 plasmid is transferred into the Escherichia coli BL21 expression strain by a conventional method, followed by induction culture and purification to obtain a recombinant protein having the amino acid sequence in the sequence table SEQ ID NO.4.

[0023] At the same time, the 483 bp fragment recovered by enzyme digestion was connected to the pCN3 vector in the same way to construct the pTroT NF-α1 recombinant plasmid.

[0024] The above-mentioned silver pomfret TNF-α1 and its application. The silver pomfret TNF-α1 protein of the present invention can significantly promote the apoptosis of liver cancer cells. The silver pomfret TNF-α1 of the present invention can improve the ability of fish to resist bacterial infection, can be used as an immunopotentiator, and has application value in the preparation of antiviral drugs, antibacterial drugs and anti-inflammatory agents, and the prevention and treatment of fish diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the predicted analysis result of the full-length protein structure of TNF-α1 of oval pomfret;

[0026] Figure 2 This is the electrophoresis diagram of the Ni column affinity purification of the oval pomfret TNF-α1 domain recombinant protein in the embodiment of the present invention; wherein M is the standard molecular weight protein; 1 represents rTrx; 2 represents rTNF-α1; the molecular weight of rTNF-α1 is 39.0 kDa;

[0027] Figure 3 The result diagram of TNF-α1 promoting apoptosis of liver cancer cells in the embodiment of the present invention;

[0028] Figure 4 The embodiment of the present invention is a graph showing the changes in the number of bacteria in the liver, spleen and head kidney at various time points after overexpression of TNF-α1 in experimental fish;

[0029] Figure 5 The embodiment of the present invention is a graph showing the changes in the number of bacteria in the liver, spleen and head kidney at various time points after the expression of TNF-α1 was interfered in experimental fish. DETAILED DESCRIPTION

[0030] 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.

[0031] The TNF-α1 of oval pomfret, the nucleotide sequence of the TNF-α1 gene cDNA of the oval pomfret is as shown in SEQ ID NO.1, the amino acid sequence of the TNF-α1 gene of the oval pomfret is as shown in SEQ ID NO.2, the nucleotide sequence of the TNF-α1 domain gene cDNA of the oval pomfret is as shown in SEQ ID NO.3, and the amino acid sequence of the TNF-α1 domain of the oval pomfret is as shown in SEQ ID NO.4.

[0032] The method for preparing the above-mentioned oval pomfret TNF-α1 domain gene recombinant protein is carried out in the following steps:

[0033] 1. Construction of recombinant vector

[0034] RNA was extracted from the head kidney tissue of the oval pomfret, and the RNA was reverse transcribed into cDNA. The nucleotide sequence of the TNF-α1 domain gene was obtained by PCR using the cDNA as a template and the following sequences as primers. The sequences of the primers are:

[0035] TroTNF-α1-F: 5'-gatatcgccaccATGGCAGCCATCCATTTAGAAGGTAG-3';

[0036] TroTNF-α1-R: 5'-gatatcAAGTGCAAACACACCAAAGAAAGT-3'.

[0037] The specific operation steps are to clone the TNF superfamily domain (95-253 amino acids) of TroTNF-α1 according to the conventional TA cloning method. Figure 1 ) was connected to the pMD19-T vector to obtain TroTNF-α1-Tsimple.

[0038] The above recombinant plasmid was extracted, and a 483 bp fragment was recovered after digestion with EcoRV restriction endonuclease; pET-32a plasmid was extracted, and after digestion with EcoRV, the above 483 bp recovered fragment was connected with it using T4 ligase to construct a recombinant plasmid; the recombinant plasmid was verified to contain TNF-α1 by gene sequencing, and it was named pET32a-TroTNF-α1. The 483 bp fragment was connected to pCN3 in the same way to obtain the pTroTNF-α1 recombinant plasmid.

[0039] 2. Induced expression and purification of recombinant protein rTNF-α1

[0040] In order to express the recombinant protein of rTNF-α1 from ovate pomfret, we introduced pET32a-TroTNF-α1 and pET32a expressing rTrx into Escherichia coli BL21, respectively, and stored the strains at -80°C. The strains were then inoculated into LB liquid medium and cultured at 37°C until OD 600 ≈0.6. Then, the cells were induced with 0.5 mM IPTG at 30 °C for 8 h and then collected. Then, the cells were resuspended in 10 mM imidazole buffer, ultrasonically broken and lysed, and the target protein was obtained by Ni column affinity purification. The recombinant protein after purification and dialysis can be found in Figure 2 , which is consistent with the expected protein size. That is, the oval pomfret rTNF-α1 recombinant protein was obtained, and the amino acid sequence is shown in SEQ ID NO.4.

[0041] Length: 160 amino acids

[0042] Type:Amino Acid

[0043] Chain type: Single chain

[0044] Characteristics: Molecular weight is 39.0kDa and isoelectric point is 4.59.

[0045] Example 1: Recombinant protein rTNF-α1 can significantly promote apoptosis of liver cancer cells

[0046] Hep G2 (liver cancer cell) cells were seeded in 6-well plates (1×10 6 Cells / well) were added and cultured at 37°C for 12 hours. They were then treated with 50 μg / mL rTNF-α1, 50 μg / mL rTrx or PBS (as a control group). After 24 hours of treatment, the cells were apoptotically detected using the Annexin V-FITC / PI cell apoptosis detection kit (BD Pharmingen TM , USA) for operation. Finally, flow cytometry was used to count the cells. FlowJo V10 was used for data analysis.

[0047] The results showed that compared with the control group, the apoptotic effect of cells treated with rTNF-α1 was significantly increased (12.6% and 39.6%, respectively; Figure 3 However, the apoptotic effect of HepG2 cells treated with rTrx (rTrx is a control protein, and the empty vector carries a tag protein Trx) was almost the same as that of the control cells (15.76%; Figure 3 ). These results indicate that TNF-α1 in Oval Pomfret can promote apoptosis of tumor cells.

[0048] Experimental Example 2: The TNF-α1 of the present invention can significantly improve the anti-bacterial infection ability of the ovate pomfret

[0049] To investigate the antibacterial immunity induced by TroTNF-α1 overexpression in vivo, we extracted the pTroTNF-α1 endotoxin-free plasmid using an endotoxin-free plasmid extraction kit (Tiangen, China) and diluted it to 200 μg / mL with PBS. In addition, 60 oval pomfret were divided into three groups (20 fish / group) and injected intramuscularly with 100 μL of pTroTNF-α1, pCN3, or PBS (as a control group). Five days after immunization, five fish were randomly selected from each group and aseptically dissected to collect the head and kidney tissues. Then, RNA from the head and kidney tissues was extracted and reversed into cDNA, which was used as a template for PCR detection to confirm the expression of the plasmid. Subsequently, the remaining fish were intraperitoneally injected with 100 μL of Vibrio harveyi (2 × 10 6 CFU / mL). At 6, 9 and 12 hours after injection, the liver, spleen and head kidney of 5 fish in each group were collected aseptically. Finally, the extracted tissues were ground, diluted and spread on LB solid medium with ampicillin. The bacterial load of each group was counted by plate count method.

[0050] In order to further determine whether TroTNF-α1 is involved in the antibacterial effect of oval pomfret, we used siRNA (small interfering RNA) technology to interfere with the expression of TroTNF-α1 in fish to see whether the antibacterial effect is weakened. TM The primers for synthesizing siTroTNF-α1, namely siTroTNF-α1-P1 / siTroTNF-α1-P2 and siTroTNF-α1-P3 / siTroTNF-α1-P4, were designed and synthesized using the Express RNAi System (Promega, USA), and siTroTNF-α1 was subsequently prepared. We randomly divided 60 fish into three groups (20 fish / group) and injected them intramuscularly with 15μg siTroTNF-α1, 15μg siTroTNF-α1-C (control siRNA) and 100μL PBS (control group). Twelve hours after siRNA injection, 5 fish were extracted from each group, and TroTNF-α1 was confirmed to be knocked down as described above. Then, 45 fish were intraperitoneally injected with 100μL of Vibrio harveyi (2×10 6 CFU / mL). 6, 12 and 24 h after injection, the bacterial load in the liver, spleen and head kidney of each group of fish was analyzed using the plate count method as described above.

[0051] The results showed that overexpression of pTroTNF-α1 could effectively reduce the bacterial load in fish (e.g. Figure 4 ). Liver: 6, 9 and 12 hours after infection, the bacterial load in fish injected with pTroTNF-α1 was about 9.5 times, 7.9 times and 3.0 times lower than that in the control group, respectively (e.g. Figure 4 ). Spleen: 6, 9 and 12 hours after infection, the bacterial load in fish injected with pTroTNF-α1 was about 2.3 times, 2.4 times and 3.2 times lower than that in the control group (such as Figure 4 ). Head kidney: 6, 9 and 12 hours after infection, the number of bacteria in fish injected with pTroTNF-α1 was about 6.2 times, 4.4 times and 2.4 times lower than that in the control group (such as Figure 4 Consistent with this, the bacterial load in the fish body was significantly increased in the three examined tissues at each time point after interference with siTroTNF-α1 compared with the control group (e.g. Figure 5 In contrast, the bacterial loads of fish treated with siTroTNF-α1-C were almost the same as those of the control group (e.g. Figure 5 ). These results indicate that TNF-α1 of ovate pomfret can enhance the body's antibacterial function.

Claims

1. Application of the TNF domain recombinant protein of TNF-α1 of silver pomfret, characterized in that: The application is to use the recombinant protein to prepare a preparation for promoting apoptosis of liver cancer cells Hep G2, and the sequence of the recombinant protein is SEQ ID NO.

4.

2. Application of the TNF domain gene of TNF-α1 of silver pomfret, characterized in that: The application is to use the TNF domain gene to prepare pTroTNF-α1 The injection preparation against Vibrio ovalis, wherein the nucleotide sequence of the TNF domain gene is SEQ ID NO.3; pTroTNF-α1 The preparation method is to obtain the nucleotide sequence of the TNF-α1 domain gene, connect it with the pMD19-T vector, transform it into Escherichia coli DH5α, sequence and identify the recombinant, and name it TroTNF-α1-Tsimple after the sequencing is correct; extract the DNA of the TroTNF-α1-Tsimple plasmid, and use Eco After digestion with RV restriction endonuclease, a 483 bp fragment was recovered, and the 483 bp fragment was ligated to the pCN3 vector to construct the pTroTNF-α1 recombinant plasmid. The DNA of the pTroTNF-α1 recombinant plasmid was extracted. pTroTNF-α1 .

Images