阅读说明：本技术 一种金钱鱼促生殖细胞成熟基因igf3及其应用 (Pseudosciaena crocea growth promoting cell maturation gene IGF3 and application thereof ) 是由 陈华谱 李智渊 李水生 张勇 江东能 田昌绪 蒋谋炎 朱春华 李广丽 于 2020-11-26 设计创作，主要内容包括：本发明公开了一种金钱鱼促生殖细胞成熟基因IGF3,它的核苷酸序列如SEQ IDNO：3所示。还公开了编码所述基因IGF3的蛋白,它的氨基酸序列如SEQ IDNO：4所示。以及所述金钱鱼促生殖细胞成熟基因IGF3的cDNA,它的核苷酸序列如SEQ IDNO：1所示。和编码所述基因IGF3的cDNA的蛋白,它的氨基酸序列如SEQ IDNO：2所示。以及包括所述基因IGF3的表达载体、金钱鱼IGF3重组蛋白及其制备方法,和上述基因IGF3、编码所述基因IGF3的蛋白或所述的重组蛋白在制备对金钱鱼卵母细胞具有促成熟作用的药物方面的应用,开发的IGF3重组蛋白有望应用于研发新型鱼类催熟催产制剂。(The invention discloses a pseudosciaena crocea growth promoting cell maturation gene IGF3, the nucleotide sequence of which is shown as SEQ ID NO: 3, respectively. Also discloses a protein for coding the gene IGF3, and the amino acid sequence of the protein is shown as SEQ ID NO: 4, respectively. And the cDNA of the pseudosciaena crocea germ-promoting cell maturation gene IGF3, the nucleotide sequence of which is shown in SEQ ID NO: 1 is shown. And a protein of cDNA encoding the gene IGF3, the amino acid sequence of which is shown in SEQ ID NO: 2, respectively. The expression vector comprising the gene IGF3, the scatophagus argus IGF3 recombinant protein and the preparation method thereof, and the application of the gene IGF3, the protein encoding the gene IGF3 or the recombinant protein in the preparation of the medicine with the maturation promoting effect on the scatophagus argus oocytes, and the developed IGF3 recombinant protein is expected to be applied to the development of novel fish maturation promoting and spawning promoting preparations.)

1. A plecoglossus altivelis germ-promoting cell maturation gene IGF3, which is characterized in that: the nucleotide sequence is shown as SEQ ID NO: 3, respectively.

2. The protein encoding the gene IGF3 of claim 1, which is characterized in that: the amino acid sequence is shown as SEQ ID NO: 4, respectively.

3. The cDNA of the Pseudosciaena crocea germ-promoting cell maturation gene IGF3 according to claim 1, which is characterized in that: the nucleotide sequence is shown as SEQ ID NO: 1 is shown.

4. A protein encoding the cDNA of the gene IGF3 of claim 3, which is characterized in that: the amino acid sequence is shown as SEQ ID NO: 2, respectively.

5. An expression vector comprising the gene IGF3 of claim 1.

6. A preparation method of a scatophagus argus IGF3 recombinant protein is characterized by comprising the following steps: comprising transforming a host cell with the expression vector of claim 5, and culturing the transformant to obtain a recombinant scatophagus argus IGF3 recombinant protein.

7. A scatophagus argus IGF3 recombinant protein, which is characterized in that: comprising the steps of transforming a host cell with the esterase expression vector of claim 5, culturing the transformant, and obtaining a recombinant protein from the culture.

8. Use of the gene IGF3 of claim 1, a protein encoding the gene IGF3 of claim 1 or the recombinant protein of claim 7 for the preparation of a medicament having a pro-maturation effect on the oocytes of scatophagus argus.

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a pseudosciaena crocea growth promoting cell maturation gene IGF3 and application thereof.

Technical Field

Reproduction and growth are the most fundamental characteristics of organisms; they are closely related but distinct from each other. Insulin-like growth factors (IGFs) are one of the key factors regulating the growth and reproductive axis. IGFs are composed of 3 ligands (IGF1, IGF2, and the latest discovered IGF3), 2 receptors (IGF1R, IGF2R), and 6 IGF-binding proteins (IGFBPs). In fish, IGF1 and IGF2 mainly exert physiological functions of promoting growth, but since the discovery of specifically expressed IGF3 in fish ovary, the role of IGFs in fish gonadal development has received extensive attention.

IGF3 is a fish-specific IGF type that was first identified clonally in zebrafish and tilapia and was found to be specifically expressed in the gonads of IGF 3. Subsequently, the presence of IGF3 was also confirmed in fish such as european bass, rainbow trout, spotted maigre, grass carp, medaka, southern catfish, and grouper, and it was suggested that IGF3 is involved in the development of gonads, suggesting that IGF3 has an important physiological function in the development of gonads of fish. Further research shows that the IGF3 recombinant protein can induce the maturation of zebrafish oocytes in vitro. Finally, the important physiological function of IGF3 in the process of inducing oocyte maturation is proved, and the IGF3 is a novel key factor for inducing oocyte maturation, so that a new path for researching fish ovum maturation is opened up.

Subsequently, the mechanism of IGF3 induced oocyte maturation was explored and subsequently developed. The present research finds that IGF3 and LH have significant relevance. In zebrafish studies, LH was found to positively regulate IGF3 expression, and related studies also found that LH-activated PKA pathways in tilapia similarly activate the IGF3 promoter and IGF3 expression. In addition, after the LH beta gene is knocked out in zebra fish by means of a gene knockout technology, the expression level of IGF3 is remarkably reduced, and the IGF3 recombinant protein can save the ovum from being immature caused by the LH beta gene knockout. Meanwhile, researches show that the expression mode of IGF3 in the development process of zebra fish ovary is similar to that of LH receptor, and further suggest that IGF3 is an important engagement factor for mediating LH signals to induce maturation of fish oocytes.

The Chromalus (Scatophagus argus) belongs to Perciformes, Cipangopalurus, and Cipangopalurus, commonly called "Jinzhou". As famous and precious seawater economic fish in south, the scatophagus argus is one of precious aquarium fish, and meanwhile, the scatophagus argus tastes delicious, so that the scatophagus argus is popular among consumers. In 2015, the team of the application breaks through the total artificial breeding technology of the scatophagus argus, but the actual production finds that the scatophagus argus is the same as other seawater fishes, the traditional artificial ripening accelerating effect is not obvious, and the large-scale fry breeding is greatly limited. Therefore, how to break through efficient artificial ripening and spawning is a great challenge for marine fishes. IGF3 has more convenient application space as a protein product for gene expression, and has incomparable advantages compared with the traditional hormone-induced oocyte maturation.

Disclosure of Invention

The invention aims to provide a merlonella growth-promoting cell maturation gene IGF3, a protein coded by the gene, cDNA, a protein coded by the cDNA and an expression vector containing the gene IGF 3.

The invention also aims to provide a scatophagus argus IGF3 recombinant protein and a preparation method thereof.

The last purpose of the invention is to provide the IGF3 gene, its coded protein or the application of the recombinant protein in preparing medicines with maturation promoting effect on roeblast cells of the scatophagus argus.

The first object of the present invention can be achieved by the following technical solutions: a plecoglossus altivelis germ-promoting cell maturation gene IGF3, the nucleotide sequence of which is shown in SEQ ID NO: 3, respectively.

The amino acid sequence of the protein for coding the gene IGF3 is shown as SEQ ID NO: 4, respectively.

The cDNA of the pseudosciaena crocea germ-promoting cell maturation gene IGF3 has a nucleotide sequence shown in SEQ ID NO: 1 is shown.

The cDNA sequence of the novel Insulin growth factor (Insulin-ike growth factor 3, IGF3) is cloned from the scatophagus argus by using a reverse transcription PCR technology.

The amino acid sequence of the protein of cDNA for coding the gene IGF3 is shown as SEQ ID NO: 2, respectively.

The cDNA sequence of the scatophagus argus IGF3 gene is shown as SEQ ID NO: 1, the total length is 594bp, the coded protein thereof has 198 amino acids, and the sequence is shown as SEQ ID NO: 2, the obtained merlonella growth-promoting cell maturation gene IGF3 does not contain the information peptide part in cDNA, the amplified fragment size is 507bp, and the sequence is shown as SEQ ID NO: 3, the coded protein has 169 amino acids in total and has a sequence shown as SEQ ID NO: 4, respectively.

The invention also provides an expression vector comprising the gene IGF 3.

The second object of the present invention can be achieved by the following technical solutions: a preparation method of the scatophagus argus IGF3 recombinant protein comprises the steps of transforming host cells by using the expression vector, culturing the transformant and obtaining the recombinant scatophagus argus IGF3 recombinant protein.

The invention also provides a scatophagus argus IGF3 recombinant protein, which is prepared by the method comprising the steps of transforming host cells by using the esterase expression vector, culturing the transformant and obtaining the recombinant protein from the culture.

The invention clones the growth promoting cell maturation gene IGF3 from the scatophagus argus by using a reverse transcription PCR technology, constructs a prokaryotic expression vector of IGF3, and can express a large amount of recombinant protein encoded by the growth promoting cell maturation gene IGF3 of the scatophagus argus.

The last object of the present invention can be achieved by the following technical solutions: the gene IGF3, the protein for coding the gene IGF3 or the recombinant protein are applied to the preparation of the medicine with the maturation promoting effect on the roeblast cells of the scatophagus argus.

The invention has the following advantages: the cDNA sequence of a novel insulin-like growth factor (IGF 3) is cloned from scatophagus argus by using a reverse transcription PCR technology, and the experimental result shows that IGF3 may participate in the physiological process of regulating and controlling maturation of scatophagus argus oocytes, so that a certain theoretical basis and important clues are provided for further understanding the reproductive endocrine regulation and control mechanism of maturation of fish oocytes. The developed scatophagus argus IGF3 recombinant protein is expected to be applied to the development of novel fish ripening and spawning-inducing preparations.

Drawings

FIG. 1 is the electrophoresis chart of PCR amplified scatophagus argus IGF3 gene in example 1;

fig. 2 shows the expression and purification of the recombinant protein of cheiliaris IGF3 in example 1, M: marker, 1: before column passing, 2: flow-through, 3-6: imidazole eluent washing at 50mM concentration, 7-10: eluting with 250mM imidazole eluent;

FIG. 3 is SDS-PAGE detection of the scatophagus argus IGF3 recombinant protein in example 1, M: marker, 1-2: washing liquid, 3-4: eluting the solution;

FIG. 4 is the mass spectrum result of the recombinant protein IGF3 of the scatophagus argus in example 2, wherein the reliability of the underlined part is more than 95%, the bold part is considered as completely unreliable and completely neglected, the coverage rate of the mass spectrum result of the recombinant protein IGF3 is 84.02%, and the recombinant protein is determined to be the target protein;

FIG. 5 shows the regulation of the expression of cell cycle genes at the mRNA transcription level by various concentrations of the recombinant Pseudolysimachia christinae IGF3 protein in example 3, A shows the effect of the recombinant IGF3 protein on cdc2 expression, and B shows the effect of the recombinant IGF3 protein on cyclinB expression.

Detailed description of the preferred embodiment

Example 1 preparation of recombinant protein of IGF3

1. Extraction of RNA from scatophagus argus

Taking healthy whole scatophagus argus, performing ice bath anesthesia for about 2min, killing the scatophagus argus, sampling, separating ovarian tissues, and extracting by using a Trizol reagent method to obtain total RNA of the scatophagus argus ovarian tissues.

2. Cloning of IGF3 Gene

First strand cDNA Synthesis:

PrimeScript from TaKaRa was used^TMAn RT reagent Kit with gDNA Eraser (Perfect Real Time) Kit, genome DNA in total RNA of the scatophagus argus ovary tissue is removed, then the total RNA is reversely transcribed to obtain single-stranded cDNA of a first chain, and the single-stranded cDNA is stored in a refrigerator at the temperature of minus 20 ℃ for later use.

Cloning of IGF3 Gene with cDNA as template:

according to the results of the related sequence alignment and structural analysis in bioinformatics, the sequence encoding a signal peptide of 29 amino acids was deleted from the cDNA sequence of the gene IGF3 (shown in SEQ ID NO: 1, and the protein sequence encoded by it is shown in SEQ ID NO: 2), and specific primers were designed at both ends with the remaining sequence as follows:

IGF3-RT-F：5’-tgggatccggtaccaagcttGCCTGGCCGCTCTCCTCTGA-3’(SEQ ID NO：5)

IGF3-RT-R：5’-tggtggtggtggtgctcgagTTATGATTCAAATCCCATCATTC-3’(SEQ ID NO：6)

the first strand cDNA is taken as a template, primers IGF3-RT-F and IGF3-RT-R are adopted for PCR amplification to obtain an IGF3 gene without information peptide, the size of an amplified fragment is 507bp, and as shown in figure 1, the amplification sequence is shown as SEQ ID NO: 3, and the coded protein sequence is shown as SEQ ID NO: 4, respectively. Separating DNA fragments by electrophoresis, cutting gel and recovering the target product. Connecting the purified target product to a pMBP vector, transforming Escherichia coli DH5 alpha, selecting positive clones for sequencing, carrying out BLAST comparison analysis on sequencing results, and determining that the target product is gene IGF3 according to comparison results.

3. Construction of prokaryotic expression vector of gene IGF3

Selecting positive cloned escherichia coli DH5 alpha, extracting plasmids by using a plasmid miniprep kit, then transforming the plasmids to prokaryotic expression engineering bacteria rosetta2, carrying out bacteria liquid PCR verification on the obtained positive monoclonal bacterial colony, wherein the size of the PCR amplified fragment is similar to that of a target fragment and is about 507bp, and carrying out sequencing verification on the positive monoclonal bacterial colony, wherein the sequencing result is consistent with the sequence of the target fragment, which indicates that the prokaryotic recombinant expression vector of IGF3 is successfully constructed.

4. Prokaryotic expression and purification of gene IGF3

Induced expression of recombinant protein:

the engineering bacterium rosetta2 containing the recombinant expression vector (pMBP-IGF3) is subjected to amplification culture in LB culture medium, and when the bacterium solution OD is obtained₆₀₀And when the value is between 0.6 and 0.8, adding IPTG (0.5mM, 37 ℃ and 4 hours) for induction culture, collecting bacterial liquid after the induction culture, centrifuging to obtain thalli, adding 10mM Tris-HCl (pH 8.0) solution containing 0.5M sodium chloride for resuspension of the thalli, carrying out ultrasonic crushing on the thalli resuspension solution, then adding protein Loading buffer, mixing uniformly, boiling in a boiling water bath for 5 minutes, and carrying out SDS-PAGE electrophoresis detection.

Purifying, desalting, enzyme cutting and freeze-drying the recombinant protein:

and (3) ultrasonically crushing the thalli after induction expression, centrifuging, discarding the precipitate, and filtering the supernatant through a filter membrane with the pore diameter of 0.45 micrometer for later use.

The nickel column was washed with deionized water to pH 7.0, and about 50mL of a 10mM Tris-HCl (pH 8.0) solution containing 0.5M sodium chloride was added to the column to equilibrate the nickel column, and the diluted sample solution was passed through the nickel column, collected as a permeate, and then passed through the nickel column twice more. After the recombinant protein is hung on the column, the column is rinsed by using 10mM Tris-HCl (pH 8.0) rinsing solution containing 0.5M sodium chloride, then imidazole eluents with the concentrations of 50mM and 250mM are respectively used for elution, and SDS-PAGE (figure 2) detection is carried out on substances eluted by the imidazole eluents with different concentrations to obtain the purified recombinant protein.

The purified recombinant protein was desalted, washed with 0.02M PB buffer and the column was equilibrated. The sample was added and the flow-through was collected. And adding a PB buffer solution for elution after the sample completely enters the column at the lower end, and collecting the eluent, namely the desalted recombinant protein.

And adding tev protease into the desalted recombinant protein for enzyme digestion, carrying out enzyme digestion at the temperature of 4 ℃ overnight according to the mass ratio of 10: 1(MBP-IGF 3: MBP-tev), and then carrying out electrophoresis to detect the enzyme digestion efficiency. After the enzyme cutting efficiency exceeds 90%, through amylose resin, the recombinant protein flowing through is collected and detected by SDS-PAGE, and the molecular weight of the IGF3 recombinant protein is about 19kDa (FIG. 3).

Freezing and storing the enzyme-cut recombinant protein: the collected liquid containing the target protein is put into a clean tube with proper size, sealed by a sealing film, and a plurality of needle holes are punched on the film so as to facilitate the water loss during freezing. The tube is vertically placed in a refrigerator with the temperature of-20 ℃ for more than about 2 hours, and is quickly placed in a refrigerator with the temperature of-80 ℃ after being completely frozen into ice. The next day, the tubes stored at-80 ℃ are placed in the frozen bottles of the frozen machine which is well debugged, and then frozen. The freezing time depends on the water loss speed, and is generally more than 8 hours or overnight. The frozen tube containing the target protein is taken down and stored in a-80 ℃ refrigerator for the next experiment.

Example 2 Mass Spectrometry assay of IGF3 recombinant proteins

Firstly, the reduction and alkylation of protein are carried out, Dithiothreitol (DTT) with the final concentration of 10mM is added to reduce IGF3 recombinant protein, Iodoacetamide (IAM) with the final concentration of 55mM is added, and finally 1 mu g of Trypsin enzyme is added for enzymolysis for 8-16 h overnight. The polypeptides produced by the enzymatic hydrolysis were desalted using a C18 column, and the desalted polypeptides were drained and then dissolved in 15. mu.L of Loading Buffer (0.1% formic acid, 3% acetonitrile). The polypeptide was analyzed on an LC-MS/MS (ekspertTMnanLC; AB Sciex TripleTOF 5600-plus) instrument, and the results were evaluated. After the LC-MS/MS is taken off the phone, the original data is directly submitted to the AB SCIEX Triple TOF^TMDatabase search was performed in the Proteinpilot software coupled to the 5600plus mass spectrometer.

The mass spectrum results (as shown in FIG. 4) showed that the confidence of the underlined part was 95% or more, and the bold part was regarded as completely unreliable and completely ignored. However, if the sequence is at the-COOH terminal, even the bold-typed part is not found to be erroneous because-COOH is very easily filtered out by LC-MS/MS.

FIG. 4 shows that the reliability of the mass spectrum result of the IGF3 recombinant protein is 84.02% with a partial coverage of 95% or more, and the protein is determined to be the target protein.

Example 3 modulation of expression of germ cell maturation regulatory genes by IGF3 recombinant proteins

The method takes the scatophagus argus purchased from the market of Zhanjiang as a research object, takes ovarian tissue specifically expressing a gene IGF3 as a material, incubates ovarian fragments with IGF3 recombinant protein in vitro, and researches the regulation condition of the IGF3 recombinant protein on the expression of related germ cell maturation regulatory genes cdc2 and cyclinB at the mRNA transcription level.

Ovarian fragments were incubated with recombinant IGF3 proteins (prepared in example 1) at concentrations of 0, 1, 100 and 10000nM for 3h, and then the expression of the germ cell maturation regulatory gene cdc2 and cyclinB at the mRNA transcription level was detected by real-time fluorescence quantification, respectively, and the results are shown in FIG. 5. The CD 2 and cyclinB gene sequences of the scatophagus argus are obtained from a transcriptome database, and then primers are designed for real-time fluorescence quantification experiments.

From the test results in fig. 5, it can be known that IGF3 recombinant proteins with different concentrations play a promoting role in cdc2 and cyclinB, indicating that IGF3 recombinant protein has a significant maturation promoting role in the oocytes of the scatophagus argus.

The present invention is described in detail by the embodiments, which should be pointed out in the above description, but the embodiments are only used for further explanation of the invention, and do not represent the scope of the invention, and other non-essential modifications and adjustments made according to the teachings of the present invention still belong to the scope of the invention.

Sequence listing

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