NL2035702B1 - Trachinotus ovatus b cell lymphoma-2 gene, trachinotus ovatus b cell lymphoma-2 protein, plasmid, and application of trachinotus ovatus b cell lymphoma-2 gene - Google Patents
Trachinotus ovatus b cell lymphoma-2 gene, trachinotus ovatus b cell lymphoma-2 protein, plasmid, and application of trachinotus ovatus b cell lymphoma-2 gene Download PDFInfo
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- A61K39/0011—Cancer antigens
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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Abstract
UITTREKSEL The present invention relates to a Trachinotus ovatus B cell lymphoma—2 gene, a Trachinotus ovatus B cell lymphoma—2 protein, a plasmid and an application of the Trachinotus ovatus B cell lymphoma—2 gene, and belongs to the field of molecular biology. A 5 cDNA nucleotide sequence of the Trachinotus ovatus B cell lymphoma—2 is shown as SEQ ID No.1, and an amino acid sequence is shown as SEQ ID No.2. The present invention also provides an application of the Trachinotus ovatus B cell lymphoma—2 as an immunopotentiator, and a method for constructing eukaryotic lO expression plasmids of the Trachinotus ovatus B cell lymphoma—2. The eukaryotic expression. plasmids of the Trachinotus ovatus B cell lymphoma—2 can improve the disease resistance of the Trachinotus ovatus body greatly after being injected into a Trachinotus ovatus body, thus having a significant immune 15 protection effect. (+ Fig. l)
Description
P1874 /NL
TRACHINOTUS OVATUS B CELL LYMPHOMA-2 GENE, TRACHINOTUS OVATUS B
CELL LYMPHOMA-2 PROTEIN, PLASMID, AND APPLICATION OF TRACHINOTUS
OVATUS B CELL LYMPHOMA-2 GENE
The present invention relates to the field of molecular biol- ogy, in particular to a Trachinotus ovatus B cell lymphoma-2 gene (Trobcl-2}, and the application of the Trobcl-2 as an immunopoten- tiator in anti-bacterial diseases of Trachinotus ovatus.
Background technology
Trachinotus ovatus, one of the rare marine fish in southern
China, has high economic value. With the deterioration of culture environment and the increase of culture density, the Trachinotus ovatus continue being suffered from diseases, resulting in huge economic losses. Therefore, it is particularly important to im- prove the disease invasion resistance capacity from the perspec- tive of the Trachinotus ovatus itself.
Apoptosis, namely programmed cell death, plays a key role in developments and steady-state regulations of multicellular organ- isms. Bcl2 family proteins are main regulatory factors in the pro- cess of apoptosis, and the function and mechanism of the Bcl2 fam- ily proteins in the process of apoptosis have always been a focus of research. Some studies have shown that the Bcl2 family proteins not only act on mitochondria to induce apoptosis, but also partic- ipate in a variety of reactions, including the regulation of cel- lular endoplasmic reticulum Ca®’, the repair of DNA damage and the interaction with autophagy, thus regulating survival states of cells from many aspects.
Bcl2 family participates in apoptosis. According to struc- tures and functions of the Bcl2 family, members of the Bcl2 family can be divided into three categories, including multi-domain anti- apoptosis proteins, multi-domain pro-apoptosis proteins and only pro-apoptosis BH-3 proteins. Bcl2 is an anti-apoptosis member of
Bcl2 family and plays an important role in regulating apoptosis; in addition to apoptosis, Bcl2 also participates in immune regula-
tion and host immune response to pathogens. Studies have shown that Bcl2 can be used as an immune adjuvant to significantly im- prove the body's disease resistance in mammals, and it has broad application prospects; however, functions and application poten- tials of Bcl2 in fish are unclear.
An objective of the present invention is to provide a Trachi- notus ovatus B cell lymphoma-2 gene, a Trachinotus ovatus B cell lymphoma-2 protein, a vector and an application of Trachinotus ovatus B cell lymphoma-2 gene in anti-bacterial drug, so as to find a new prevention and control strategy for aquatic animal dis- eases and promote the healthy and sustainable development of aqua- culture in China.
The present invention is achieved by the following technical solutions:
A Trachinotus ovatus B cell lymphoma-2 (TroBcl2) gene, with a nucleotide sequence shown as SEQ ID NO.1: atggcgagcgagtgtaatcgcaacatcgtggaaaagtatatctgcca- taaactctccaaacgggggtacgtgtggggatttgatgacgtccgggatgaagacgctgctaa- taatggctcaatagttgcccctccaccgactctggtccgcceggtgccgtgaagccagcac- cgggcctgacaacgaggacgcatccaacctgtgcagacggctcecgcagtecgacccgcac- gcegccatccacagagtcctgcgtgaggcecggagacgaacttgaaaggttgtaccagccg- gacttcacggagatgtcgcggcagctctatctcacctectccacggcgcagaggagat- tcgccgaggtgatagacgagctgttcegggacggagtgaactggggccggat- tatcgcgttecttcgagtteggggggaecggtgtgtgctgagtgcgtggccaaagagga- gatgacatcgcaggtggacaacatcgcggagtggatgacggagtatttaaatggac- ctcttaacagctggataaaggataacgggggaetgggatgcctttgtggagctgtatgataga- cagagggagtccatcttcagttgctectggccctccatcaagacggtcttcggtctggetg- cactcggggcagctagcctcaccatcggagcgtaccttacacagaagtga.
A Trachinotus ovatus B cell lymphoma-2 protein encoded by the
Trachinotus ovatus B cell lymphoma-2 gene, with an amino acid se- quence shown as SEQ ID NO.Z:
MASECNRNIVEKYICHKLSKRGYVWGEDDVRDEDAANNGSIVAPPPTLVRRCREAST-
GPDNEDASNLCRRLPOSDPHAAIHRVLREAGDELER-
LYQPDFTEMSROLYLTSSTAQRRFAEVIDELFRDGVNWGRIIAFFEFGGTVCAE-
CVAKEEMTSOVDNIAEWMTEYLNGPLNSWIKDNGGWDAFVELYDRORESIESCSWPSIK-
TVEFGLAALGAASLTIGAYLTOK.
A recombinant vector including the Trachinotus ovatus B cell lymphoma-2 gene.
A recombinant plasmid including the recombinant vector.
The present invention also provides an application of the
Trachinotus ovatus B cell lymphoma-2 gene (TroBcl2}) in preparing an anti-bacterial infection preparation of Trachinotus ovatus.
Further, the bacterium is Vibrio harveyi.
A cloning method of the above TroBcl2 gene is as follows: to- tal RNAs from the head and kidney tissues of a Trachinotus ovatus are extracted, the RNAs are subjected to reverse transcription in- to cDNA, then the cDNA is used as a template and the following se- quences are used as primers so as to obtain a full-length sequence through PCR amplification. Sequence of the primers are as follows:
TroBcl2-F:5'’-gatatcgccaccatggcgagcgagtgtaatcgc-3’
TroBcl2-R:5' -gatatecttctgtgtaaggtacgctccgatg-3'’
PCR reaction conditions: pre-denaturing at 95°C for 5 min, at 95°C for 30 s, at 58°C for 30 s and at 72°C for 1 min for 32 cycles, and finally extending at 72°C for 5 min.
An eukaryotic expression plasmid of the Trobcl2 gene (pTroBcl2) is amplified with primers TroBcl2-F and TroBcl2-R by using Trobcl2 cDNA as a template, and the amplified fragments are ligated with pEASYS-T1 Simple vector after gel extraction, and transformed into Escherichia coli DH5a competent, and positive colonies are selected for PCR detection; a recombinant plasmid is extracted after the detection is correct, and 687bp fragments are extracted after restriction endonuclease digestion with EcoR V; an eukaryotic expression vector pCN3 plasmid is extracted and pCN3 vector is digested with Sma I, and the 687bp extracted fragments are ligated by T4 DNA ligase to construct recombinant plasmid; and the recombinant plasmid is confirmed to include Bcl2 gene by gene sequencing, and it is named pTroBcl2.
Compared with the prior art, the present invention has the following beneficial effects.
The TroBcl2 eukaryotic expression plasmid can promote expres- sions of anti-inflammatory factors such as IL-10 and TGF-p after being injected into fish, and can significantly improve survival rates of fish infected by bacteria, thus having a significant im- mune protection effect.
FIG. 1 is a detection diagram for an immuncprotective effect of the eukaryotic expression plasmid of TroBcl2 (pTroBcl2); and
FIG. 2 is an experimental diagram for an influence of pTroBcl2 on expressions of immune genes IL-10 and TGF-B, where,
FIG. A is relative expression quantities of immune genes in a spleen tissue; and FIG. B is relative expression quantities of im- mune genes in head and kidney tissues.
Specific embodiments
The technical solutions of the present invention will be fur- ther explained by the following examples, but the technical solu- tions of the present invention are not limited in any form by the examples.
Example 1: cloning of TroBcl2 gene (1) Total RNAs are extracted by a total RNA extraction kit of
Promega company. (2) Synthesis of a first strand of cDNA: the extracted total
RNAs are used as a template, and a reverse transcription kit (GoScript™ Reverse Transcription Mix, Oligo (dT), A2791) of Promega company is used to synthesize the first strand of cDNA, with the operation steps as follows: 1) a mixed solution is prepared, as shown in Table 1:
Table 1 Preparation system of a mixed solution
TT component Addtionquantity
OO TotalRNA iw
GoScript™ Enzyme Mix 1 ul
GoScript™ Reaction Buffer 4 ul
Nuclease-free water 14 uL
Total 20 pL
TZ) After being mixed, the mized solution is put into a PCR am- plifier and a reverse transcription program is set, as shown in
Table 2:
Table 2 PCR reverse transcription program
Temperature Time - s~c em 98 °C 5 min 4°C Hold 3) After completion of the program, the obtained cDNA is _ 5 stored at -20°C for later use. (3) Amplification of TroBcl 2 gene
A coding region sequence of TroBcl 2 is retrieved from the transcription group library in a laboratory, and the following primers are designed for an amplification of the TroBcl2 gene;
Forward primer TroBcl2-F:5'- gatatcgccaccatggcgagcgagtgtaatcgc-3'
Reverse primer TroBcl2-R:5'-gatatccttctgtgtaaggtacgctcegatg- 37
PCR reagents and conditions: 1) a mixed solution is prepared, as shown in Table 3:
Table 3 Preparation system of a PCR mixed solution
Component Additongquantty - TtroBo2F ix
TroBel2-R 1ul cDNA template Tul
Trans-Tag 0.2 HL 10xBuffer 25uL dNTP 0.5 ul ddH,0 18.8 ul
Total 25uL
TT 2) After being mixed, the mixed solution is put into a PCR am- plifier and a reverse transcription program is set, as shown in
Table 4:
Table 4 PCR reverse transcription program = Temperature ________ tme - esc sem 95 °C 30s 58 °C 30s 32 cycles 72°C 1 min 72°C 5 min 4°C Hold 3) After completion of the PCR reaction, 1.2% agarose gel is used to detect an amplified product, then a FastPure ® Gel DNA Ex- traction Mini Kit of Vazyme Biotech Co., Ltd. is used to purify the product, and a PCR product is extracted from the gel according to the steps of specification. (4) TA cloning: 1) vector ligation
According to instructions of pEAsY*-T1 simple Cloning Vector (TransGen Biotech), a ligation system is prepared as shown in Ta- ble 5, and incubated at room temperature for 10 min.
Table 5 Ligation system ~ Compomemt Additionguantity © pEASYST1simple Cloning Vector ipl
Bel2 glue extraction product 4 pul ddH,0 ipl
Total 6 pL ~~ 2) Transformation
DH5Qa competent cells stored at -80°C are taken out and placed on ice until they are dissolved, 6 uL of ligation products are added, and ice bath is carried out for 30 min.
Heat shock is carried out at 42°C for 90 s and ice bath is carried out for 5 min. 800 pL of LB liquid medium is added, and shaking culture is carried out at 37°C and 180 rpm for 1 h.
Centrifugation is carried out at 4000xg for 5 min, 800 pL of supernatant is discarded, and bacteria are resuspended and coated on an LB solid plate including Amp antibiotics and IPTG/X-Gal so as to screen clones, and the screened clones are cultured upside down at 37°C for 12 h. 3) Screening and detection of positive clones 10 pL of sterile ddH;O is added into a 200 pL of PCR tube, monoclones (white spots) are selected and put into the PCR tube, and then blowing and mixing well are carried out. 1 pL of ddH:0 including bacteria is used as a template, and the primer TroBcl2-
F/R is used to carry out PCR amplification, with the PCR amplifi- cation system the same as above. 1% agarose gel electrophoresis is used to detect PCR products, positive strains are cultured, pre- served and sequenced, and the correctly sequenced strains are stored at -80°C.
Example 2: construction of Trobcl 2 eukaryotic expression vector (1) Plasmid extraction:
A bacterial solution of the TroBcl2 plasmid and the eukaryot- ic expression vector pCN3 plasmid that have correct sequencing mentioned above and contain complete ORF sequence is prepared in advance, and a Plasmid Mini Kit I of Omega company is used to ex- tract the plasmids. (2) Enzymatic digestion:
The above extracted plasmids (pCN3 and TroBcl2) are digested with Sma I and EcoR V respectively, and an enzymatic digestion system is shown in Table 6:
Table 6 Enzymatic digestion system
Component Additionquantty
TO EoRV/SWal aw 10xFast Digest buffer 3 ul ddH,0 11 pl
Plasmid 15 pl total 30 ul | The prepared system is incubated in a thermostatic metal bath at 37°C for 30 min, and digestion results of the TroBcl2 plasmid can be directly detected after the TroBcl2 plasmid is digested, while digestion results of the pCN3 vector can be detected only after the enzymatic digestion system of the pCN3 vector is further incubated for 10 min with 3 pL FastAP.
(3) A FastPure ® Gel DNA Extraction Mini Kit of Vazyme Bio- tech Co.,Ltd. is used for the gel extraction of enzyme digestion product. (4) Vector ligation and transformation 1) ligation: the digested TroBcl2 and pCN3 are ligated with
T4 DNA ligase to prepare the following ligation system, and the ligation condition is 16°C overnight.
Table 7 Ligation system ~ Component Additionquantty
CO pCN3extactionproduet ia.
TroBcl2 extraction product 6 HL
T4 DNA ligase 1 aL 10xT4 DNA ligase buffer lp ddH,0 1 ul
Total 10 pL ~ 2) Transformation
DH5x competent cells stored at -80°C are taken out and placed on ice until they are dissolved, 10 pL of ligation products are added, and ice bath is carried out for 30 min.
Heat shock is carried out at 42°C for 90 s and ice bath is carried out for 5 min. 800 pL of LB liquid medium is added, and shaking culture is carried out at 37°C and 180 rpm for 1 h.
Centrifugation is carried out at 4000xg for 5 min, 800 pL of supernatant is discarded, and bacteria are resuspended and coated on an LB solid plate including Amp antibiotics, and cultured up- side down at 37°C for 12 h. 3) Screening and detection of positive clones 10 pL of sterile ddH, is added into a 200 pL of PCR tube, and monoclones are selected and put into the PCR tube, and then blowing and mixing well are carried out. 1 pL of ddH;0 including bacteria is used as a template, and the primer TroBcl2-F/CN-R and
TroBcl2-R/CN-F is used to carry out PCR amplification, with the
PCR amplification system the same as above. 1% agarose gel elec- trophoresis is used to detect PCR products, and positive strains are cultured, preserved and sequenced, and the correctly sequenced strains are stored at -80°C.
The primers are:
TroBcl2-F:5'’-gatatcgccaccatggcgagcgagtgtaatcgc-3'
TroBcl2-R:5'-gatatcecttctgtgtaaggtacgctccecgatg-3'
CN-F:5/-cttgcgtttctgataggcaccta-3/'
CN-R:5" -TGCGGGCCTCTTCGCTATT-3'
Example 3: application of pTroBcl2 (1) Extraction of endotoxin-free plasmids of pTroBcl2 and
PCN3
Endotoxin-free plasmids of pCN3 and pTroBcl2 are extracted by an endotoxin-free plasmid mass extraction kit of TIANGEN Bio- tech (Beijing) Co.,Ltd., and are stored at -20°C. (2) Plasmid injection: the endotoxin-free plasmids of pTroBcl2 and pCN3 extracted above are diluted with PBS to 150 pg/mL. 60 Trachinotus ovatus are randomly divided into three groups, with 20 in each group; three groups are intramuscularly injected with 100 pL of the above well-diluted pTroBcl2, pCN3 and
PBS (control group) respectively.
Composition of the PBS: 137 mmol/L of Nacl , 2.7 mmol/L of
KCl, 10 mmol/L of Na:HPC, and 2 mmol/L of KH,PO,, and the rest is water; with pH of 7.2-7.4. (3) Preparation of bacterial suspension
Vibrio harveyi is inoculated into an LB medium, cultured by shaking at 200 rpm/min and 30°C until ODsya is about 0.8, then di- luted in PBS until a final concentration is 10° CFU/ml, and finally a Vibrio harveyi suspension is obtained. (4) Detection of immune protection effect 15 days after plasmid injection, each one of the three groups of Trachinotus ovatus is intraperitoneally injected with 100 pL of the bacterial suspension in the above step (2). A death number of
Trachinotus ovatus in each group is continuously observed and rec- orded for 10 days. After 10 days, a total death number of Trachi- notus ovatus in each group is counted: pTroBcl2 group: 5; pCN3 group: 10; PBS group: 11; and the survival rates of Trachinotus ovatus in pTroBcl2 group, pCN3 group and PBS group are 75%, 50% and 45% respectively. The survival rate of Trachinotus ovatus im-
munized with pTroBcl2 is significantly higher than that of control group and that of pCN3 group.
These results show that pTroBcl2 is an efficient immunopoten- tiator, can significantly enhance the capacity of Trachinotus ovatus to resist bacterial infection, and has a good immune pro- tection effect.
Example 4: effect of pTroBcl? on expressions of IL-10 and
TGF-P (1) Plasmid injection: the endotoxin-free plasmids of pTroBcl2 and pCN3 extracted in Example 3 are diluted with PBS to 150 pg/mL. 15 Trachinotus ovatus are randomly divided into three groups, with 5 in each group; the three groups are intramuscularly injected with 100 pL of the above well-diluted pTroBcl2, pCN3 and
PBS respectively. (2) Detection of gene expression
On the 15th day after the injection in the above step (1), head, kidney and spleen tissues of the three groups of Trachinotus ovatus are taken, and RNAs are extracted and reversed into cDNA for gRT-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) is involved in the antibacterial immune re- sponse. Developmental and Comparative Immunology, 2022, 133: 104445) to detect the expressions of IL-10 and TGF-f. The results show that the expressions of anti-inflammatory factors IL-10 and
TGF-B in head, kidney and spleen tissues of Trachinotus ovatus in pTroBcl2 group are significantly higher than those in control group (P<0.05); however, the expressions of IL-10 and TGF-B in head, kidney and spleen tissues of Trachinotus ovatus in pCN3 group are not significantly different from those in control group (P>0.05).
These results indicate that the plasmid pTroBcl2 including
TroBcl2 gene can significantly enhance the expressions of IL-10 and TGF-B in Trachinotus ovatus.
Claims (6)
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