CN110776570A

CN110776570A - Recombinant transposase and application thereof

Info

Publication number: CN110776570A
Application number: CN201911008446.8A
Authority: CN
Inventors: 邹曙明; 刘子茵; 郑国栋; 陈杰
Original assignee: Shanghai Ocean University
Current assignee: Shanghai Ocean University
Priority date: 2019-10-22
Filing date: 2019-10-22
Publication date: 2020-02-11

Abstract

The invention discloses a recombinant transposase which contains a Tgf2 sequence and an SV40NLS sequence, wherein the Tgf2 sequence is positioned at the upstream of the SV40NLS sequence, and the recombinant transposase can be efficiently positioned in the nucleus of zebra fish, so that the activity and the efficiency of the transposase are remarkably improved.

Description

Recombinant transposase and application thereof

Technical Field

The invention belongs to the field of aquatic science, relates to application of a Tgf2 transposition element, and particularly relates to an insertional mutagenesis research of a goldfish Tgf2 transposition element.

Background

Transposons, a type of DNA fragment that is capable of freely jumping (replicating or translocating) on the genome, the process of which is called transposition. Obtained by Clintock, B.M for the first time in 1950 in maize chromosomes and named "Transposable elements". Its discovery has improved the long-standing view of maintaining genomic sequence stability, and has also broken the theory of the position fixation of genetic material on chromosome. Since the research researchers have confirmed that the transposon system is widely existed in many fields of nature, including various organisms such as bacteria, fungi and insects, the research of transposon system has become an important tool for the gene analysis of organisms. By using the unique transposition characteristics of transposon elements, the marked transposon elements can be inserted into a target gene or a target genome, and then transposon tagging, transposon site-directed hybridization, transposon gene targeting and other technologies are formed. By applying the above techniques, the difference in functions of genome and between groups can be explored, the activity of the target gene can be changed, the transgenic organism can be obtained, the gene integration can be accelerated, and the gene therapy can be realized.

As a gene tool, the currently more popular transposon genes are being studied from four transposon-producing families: tcl/mariner, hAT, PIF/Harbinger and PiggyBac (PB) transposon families ^[1]. Despite the widespread distribution of transposons in nature, the long-term evolution process has led to the loss of transposition activity in most mammalian and avian transposon genomes due to the gradual inactivation of transposase, which has led to the development and application of vertebrate transposons in a slow-growing stage ^[2-3]. Until Koga et al found Tol2 transposon in medaka genome with autonomous transposition activity, they triggered the entire academic community's enthusiasm for transposon research. The Tol2 transposon belongs to a naturally occurring vertebrate transposon ^[4]Its discovery has facilitated research on the use of transposons in vertebrates.

The hAT transposon family, a naturally occurring superfamily with autonomous transposition activity, includes the Ac/Ds transposition system in maize, and the Tol2 transposon found in fish.

Goldfish Tgf2(GenBank: HM146132) transposon elements were obtained from screening in 19 different fish species by the Syasu laboratory, and are DNA transposons with autonomous activity belonging to the hAT transposon family. The total length of the transposase protein is 4720bp, has 4 ORFs, and can encode the transposase protein with the total length of 686 amino acids. The goldfish Tgf2 transposon has the characteristics of the hAT transposon family, in which the inverted repeats of the meta-position can form a cross structure different from the Tol2 transposon, the transfer of which with Tgf2Seat activity is related to ^[5]。

It is known that all substances entering and exiting the nucleus by means of passive transport and active transport occur via Nuclear Pore Complexes (NPCs) on the nuclear membrane. The structure and function of NPC appears to be conserved in all eukaryotes, suggesting its central role in eukaryotic cell function. As the name implies, NPC has the function of a porous molecular sieve, i.e., molecules with molecular weights of less than 40-45kDa can diffuse freely into and out of the nucleus of the cell. Proteins larger than 45kDa require a Nuclear Localization Signal (NLS) to aid their transport into the nucleus.

Hypoxia Inducible Factors (HIFs) are composed of an evolutionarily conserved family of transcriptional regulators that are capable of steady-state responses under low oxygen tension and play a key role in coordinating cellular responses to hypoxia ^[6]HIFs are heterodimeric complexes formed by the labile α subunit (1 α,2 α, or 3 α) and one constitutive HIF β subunit together ^[6-8]. O in the ODD Domain of the HIFs under normoxic conditions ₂Under hypoxic conditions, conserved asparagine residues in the TAD (terminal activating domain) domain are subject to hydroxylation by the inhibitor HIF-1 (FIH) under the action of the hydroxylation efficiency is reduced, they subsequently form heterologous HIF dimers with the 539392 subunit, enter the nucleus, co-factor activating regions of the CBP family such as P300/CBP family, and activate the target genes in response to hypoxia, thus degrading in combination with the E3 ubiquitin ligase protein (pVHL) targeted to the proteasome.

The structure and function of HIFs are important in explaining the molecular mechanisms of the hypoxic response, however, our studies on HIF3 α are limited compared to the intensive studies on HIF1 α and HIF2 α, and the studies on the hypoxic response of fish in particular are much less well understood.

Reference documents:

[1]Ni J,Clark K J,Fahrenkrug S C,et al.Transposon tools hopping invertebrates[J]. Briefings in Functional Genomics&Proteomics,2008,7(6):44-453.

[2]Largaespada D A,Generating and manipulating transgenic animalsusing transposable elements[J].Reproductive Biology&Endocrinology,2003,1 (1):1-10.

[3]Kempken F,Windhofer F,The hAT family:a versatile transposon groupcommon to plants,fungi,animals,and man[J]2001,110(1):1-9.

[4]KogaA,Suzuki M,Inagaki H,et al.Transposable element in fish[J].Nature,1996, 383(6595):30.

[5] cloning of the goldfish hAT family transposon Tgf2 and its structure [ J ] genetic, 2010,32(12): 1263-.

[6]Nath B,Szabo G.Hypoxia and hypoxia inducible factors:diverse rolesin liver diseases[J].Hepatology,2012,55:622-633.

[7]Semenza,G.L.,1999.Regulation of mammalian O ₂homeostasis byhypoxia- inducible factor 1[J].Annual Review ofCell andDevelopmental Biology,1999 15:551-578.

[8]Semenza,G L.HIF-1:mediator of physiological and pathophysiologicalresponses to hypoxia[J].Journal of Applied Physiology,2000,88:1474-1480.

[9] Application of Liulin, Zhang Mei, Huangyue DNA transposon in the research of mouse gene function [ J ]. heredity, 2011,33(5):485 and 493.

[10]Lampe D J,Churchill M E,Robertson H M.A purified marinertransposase is sufficient to mediate transposition in vitro[J].EMBO J,1996,15:5470–5479.

[11]Xu H L,Shen X D,Hou F,et al.Prokaryotic Expression andPurification of Soluble Goldfish Tgf2 Transposase with Transposition Activity[J].Mol Biotechnol,2015,57:94-100.

[12]Luo J C,Shibuya M.A variant of nuclear localization signal ofbipartite-type is required for the nuclear translocation of hypoxia induciblefactors(1a,2a and 3a) [J].Oncogene,2001,20:1435–1444.

[13] Xuhaili, research on prokaryotic expression, purification and DNA binding activity of goldfish Tgf2 transposase [ D ] shanghai, 2013.

Disclosure of Invention

The Tgf2 transposase protein can catalyze the breakage and recombination of DNA strands, and plays a key role in the process of functioning of the Tgf2 transposon in transposition. The precondition that the Tgf2 transposase protein can enter the nucleus is that the protein plays a role, the research focuses on improving the nucleus entering efficiency of the transposase protein and further improving the transposition efficiency of Tgf2, and the research aims to artificially interfere with a transposition element to improve the transposition efficiency, establish a theoretical basis for exogenous gene integration and stable expression and provide support for fish insertional mutagenesis research. We constructed pEGFP-Tgf2TP expression vector containing Tgf2 transposase protein and pEGFP-TP-SV40NLS transposase plasmid containing Simian virus 40(Simian virus 40, SV40) large T antigen Nuclear Localization Signal (NLS) to investigate whether artificial interference transposable element could improve Nuclear entry efficiency and transposition efficiency. The constructed transposase plasmid is introduced into fertilized eggs of zebra fish through co-microinjection, and the green fluorescent protein of the reporter gene is observed and detected at different embryonic development stages after in vitro culture, and the positive rate is detected through sequencing.

The HIFFs can activate a series of genes sensitive to Hypoxia downstream of the HIFFs to be transcribed, so that various physiological processes including energy metabolism, erythropoiesis, angiogenesis, cell proliferation and apoptosis are regulated to ensure the steady state of an organism under a hypoxic environment, the research on the structural characteristics of HIF3 α and the regulation mechanism under the hypoxic condition is less, the research on the structural characteristics and the function of HIF1 α and 2 α in humans, mammals and fish is hopeful to explore the regulation mechanism which is not Hypoxia-resistant, and further the research on the Hypoxia-resistant regulation mechanism is hopeful to be carried out by researching the structure and the Hypoxia function of HIF1 α and 2 α, so that a new strain of Megalobrama amblycephala is developed for the subsequent research on the Hypoxia-resistant regulation mechanism, and the theoretical results of the transcriptional expression of HIF 365631-EGFS 7-3 α -3 α of the expression vector containing the gene of the HIF3 α and the Hypoxia-resistant regulation mechanism of Hypoxia are provided for researching the theoretical results and the theoretical results of the Hypoxia-resistant transcriptional protein level of the Hypoxia-resistant expression vector, and the transcriptional protein level of the transcriptional gene of the Hypoxia-resistant Megalobrama:

(1) through transfecting pEGFP-Tgf2TP and pEGFP-TP-SV40NLS transposase plasmids into HeLa cells respectively, we find that green fluorescence of pEGFP-Tgf2TP-SV40NLS recombinant transposase proteins is mostly concentrated in nuclear distribution, compared with cells of a control group which are transiently transfected with pEGFP-Tgf2TP transposase plasmids, the nuclear entry efficiency reaches 51.63%, the nuclear entry efficiency is improved by 11.58% compared with the control group, and SV40NLS can help to improve the nuclear entry efficiency. The zebrafish positive rate of the microinjected pEGFP-TP-SV40NLS transposase plasmid reaches 60.8 percent, is improved by 12.7 percent compared with the positive rate of 48.1 percent of a control group, and shows that SV40NLS can help to improve the integration efficiency.

(2) The megalobrama amblycephala HIF3 α belongs to a bHLH family and has PAS, PAB, PAC, ODD and TAD structural domains which are specific to the family, and after HeLa cell transfection, the megalobrama amblycephala HIF3 α protein is concentrated in cell nucleus distribution under normal oxygen and low oxygen conditions, and the low oxygen treatment can cause that the expression level of the megalobrama HIF3 α in a detected tissue is obviously increased in mRNA and protein level.

Since the Tgf2 transposase plays an important role in the transposition process, it is necessary to conduct more intensive research on the Tgf2 transposase.

The megalobrama amblycephala is an important economic fish in China, but the megalobrama amblycephala cannot tolerate hypoxia to cause massive death caused by hypoxia in the feeding process, so the hypoxia response mechanism of the megalobrama amblycephala HIF3 α is fundamentally understood, the breeding selection can be enhanced, and the economic benefit of the production of the megalobrama amblycephala in the industry is improved.

The goldfish Tgf2 transposon belongs to the class II DNA transposon and is transposed in a "cut and paste" manner. Transposition mainly involves two processes, that is, a transposable element is cut from its original position and then inserted and integrated into genomic DNA, thereby causing gene mutation or rearrangement, and in general, the genome size is not changed. Both processes are catalyzed by the transposase encoded by the Tgf2 transposon itself.

The donor plasmid with Tgf2 transposon and the mRNA of transposase plasmid containing the transposon code are injected into the early embryo of the zebra fish, and the transposase catalyzes the transposon to play a function, so that the transposon is cut from the donor plasmid and is stably integrated into the genome of the zebra fish. Subsequently, the excessive mRNA and transposase were gradually degraded, the transposase activity was then lost, the Tgf2 transposition sequence was stably present in the genome, and the green fluorescent protein in the Tgf2 transposon plasmid helped us observe its expression with the aid of a microscope.

More specifically, in order to solve the disadvantages of the prior art, the first aspect of the present invention provides a recombinant transposase, the protein sequence of which comprises a protein sequence of Tgf2 and a protein sequence of NLS.

In some embodiments, the coding sequence of the Tgf2 protein sequence has GenBank number: HM 146132.

In some embodiments, the NLS protein sequence is an SV40NLS protein sequence.

In some embodiments, the Tgf2 protein sequence is located upstream of the NLS protein sequence.

In some embodiments, the protein sequence of Tgf2 is set forth in SEQ ID No. 1; the NLS protein sequence is shown as SEQID NO. 2.

In a second aspect, the present invention provides a recombinant transposase gene capable of encoding a recombinant transposase as described in the first aspect of the present invention.

In some embodiments, the coding sequence of gf2 protein in the recombinant transposase gene is set forth as SEQ ID number 3; the coding sequence of the NLS protein sequence is shown as SEQ ID NO. 4.

In a third aspect, the present invention provides a recombinant expression vector comprising the recombinant transposase gene according to the second aspect of the present invention.

In a fourth aspect, the invention provides the use of a recombinant transposase as described in the first aspect of the invention or a recombinant transposase gene as described in the second aspect of the invention or a recombinant expression vector as described in the third aspect of the invention for increasing the efficiency of transposition in fish.

In some embodiments, the fish is selected from the group consisting of megalobrama amblycephala, goldfish, zebrafish.

Drawings

FIG. 1 is an electropherogram of a fragment of Tgf2 TP. Transposase sequences were obtained using marker III.

FIG. 2 is an electropherogram of a fragment of Tgf2 TP. The transposase sequence obtained by PCR amplification of the ligation product PMD-19T-Tgf2TP was used with marker III.

FIG. 3 is an electrophoretogram of the double cleavage results of PMD-19T-Tgf2TP and pEGFP-N1 plasmids, which contain markerIII and D15000.

FIG. 4 is an electropherogram of transposase sequences obtained by PCR amplification of ligation products using marker III.

FIG. 5 is a diagram showing the structure of pEGFP-Tgf2TP plasmid, wherein pEGFP-Tgf2TP is a fusion protein plasmid constructed using pEGFP-N1 as a vector.

FIG. 6 is a diagram showing the electrophoresis pattern of the double cleavage result of pEGFP-Tgf2TP plasmid.

FIG. 7 shows a transposase sequence obtained by PCR amplification of the ligation product using markerIII, with Tgf2TP-SV40 NLS.

FIG. 8 is a structural diagram of plasmid pEGFP-TP-SV40NLS, wherein the Tgf2 transposase fusion protein plasmid pEGFP-TP-SV40NLS is constructed by using pEGFP-N1 as a vector.

FIG. 9 is a photograph of green fluorescence of zebrafish in a negative control group, which was injected with DNA of donor plasmid pTgf2-EF1 α -eGFP alone, and observed under white light for A/B/C, fluorescence under blue light for D/E/F, and 12h, 24h, and membrane-out period for A/D, B/E, C/F, respectively, after microinjection.

FIG. 10 is a green fluorescence photograph of zebra fish of the positive control group; this control group was injected with donor plasmid DNA and constructed pEGFP-Tgf2TP transposase plasmid mRNA, A/B/C: observed under white light, D/E/F: fluorescence observation under blue light. A/D, B/E, C/F are 12h, 24h after microinjection and membrane emergence period, respectively.

FIG. 11 is a fluorescent green photograph of zebra fish in the experimental group; this experimental group was injected with donor plasmid DNA and constructed pEGFP-TP-SV40NLS mRNA, A/B/C: observed under white light, D/E/F: fluorescence observation under blue light. A/D, B/E, C/F are 12h, 24h after microinjection and membrane emergence period, respectively.

FIG. 12 shows the detection of exogenous gene GFP in zebra fish.

A: a negative control group; b: a positive control group; c: pEGFP-TP-SV40NLS experimental group.

The Marker used for the electrophoresis detection is D2000, 1-18 are respectively detected samples, and the target band is 486 bp.

FIG. 13 is a photograph of HeLa cell transfection experiment;

A/B/C, only pEGFP-N1 plasmid is transfected; D/E/F, transfecting a pEGFP-Tgf2TP plasmid; G/H/I, pEGFP-TP-SV40NLS plasmid is transfected. A/D/G is the distribution of GFP in HeLa cells; B/E/H nuclear DAPI staining; C/F/I fusion pictures of GFP and nuclear distribution. The scale bar is 100 μm.

FIG. 14 is a statistical chart of transfection results of HeLa cells;

only transfecting pEGFP-N1 plasmid; b, transfecting a pEGFP-Tgf2TP plasmid; and C, transfecting pEGFP-TP-SV40NLS plasmid.

Detailed Description

In order to better explain the technical scheme of the invention, the following detailed description of the embodiment of the invention is combined with the accompanying drawings. The following examples are intended to further illustrate the invention but should not be construed as being limitations or restrictive thereon. Unless otherwise specified, technical features used in the embodiments may be replaced with other technical features known in the art having equivalent or similar functions or effects without departing from the inventive concept.

Example 1: insertional mutagenesis study of transposable element of goldfish Tgf2

1.1 Experimental materials, Main instruments, chemical and biological reagents

1.1.1 Experimental animals and materials

The zebra fish breeding system used in the experiment is purchased from Shanghai Shengshui equipment factories, a 14h bright/10 h dark alternating illumination system is adopted, the temperature is controlled at 28.5 ℃, the relative humidity is kept at 75-85%, and the pH value of the water body is maintained at 7.2-7.4.

The subject goldfish Tgf2 transposase sequence of this study was amplified from the pGE-4T-1-Tgf2TP plasmid ^[11-12]The plasmid was constructed in this laboratory and stored in an ultra-low temperature freezer at-80 ℃.

pEGFP-N1 plasmid (GenBank accession #: U55763) was purchased from Clontech. The plasmid contains a green fluorescent protein (EGFP) marker with a CMV promoter, and the resistance is Kana resistance (Kan) ⁺) And storing in an ultra-low temperature refrigerator at-80 ℃.

HeLa cells, purchased from CCTCC (Wuhan), used in this experiment were adherent cells commonly used for transient transfection of cells.

1.1.2 Main Instrument

The main instruments used in this experiment are shown in the following table (table 1):

experimental Equipment and manufacturer used in Table 1

1.1.3 Primary reagents

The main instruments used in this experiment are shown in the following Table (Table 2)

Chemical and biological reagents used in Table 2

1.2 Experimental methods

1.2.1 reagent formulation

1) Ampicillin/kanamycin (Ampicillin, Amp; kanamycin, Kan): 250mg of each ampicillin and kanamycin powder is dissolved in 25mL of ultrapure water (0.1 g/. mu.L), subpackaged when the dissolution is complete, and stored in a refrigerator at-20 ℃ for standby. 2) LB liquid medium: 6.25g of LB powder was added to 250mL of ultrapure water, autoclaved, and stored in a refrigerator at 4 ℃ for future use. 3) LB solid medium: 10g of LB powder is weighed into an Erlenmeyer flask, then 250mL of ultrapure water is added, after autoclaving, the mixture is naturally cooled to about 50 ℃ and the desired resistant solution is added and mixed. Pouring into a sterilized flat plate quickly, pouring the flat plate into an incubator at 37 ℃ for standing overnight after natural solidification, packaging, marking and storing for later use. 4) Media (Dulbecco's modified eagle medium, DMEM.) recipe: m199 culture medium 500mL + 10% fetal bovine serum + 1% double antibody (ampicillin/kanamycin), mixing, and placing in 4 ℃ refrigerator for use.

1.2.2 obtaining of Tgf2 transposase cDNA fragment

1.2.2.1 primer design

The Tgf2 transposase protein is derived from pGE-4T-1-Tgf2TP recombinant plasmid preserved in laboratory, and an upstream primer TP-F1 and a downstream primer TP-R1 for amplifying Tgf2TP are designed according to the sequence of the recombinant plasmid. Since the cleavage sites at both ends of Tgf2TP in the template plasmid pGE-4T-1-Tgf2TP are inconsistent with those in the subsequent experiments, the required XhoI and BamHI cleavage sites (underlined) are added when the upstream and downstream primers are synthesized, and a target fragment containing the required cleavage sites, namely the Tgf2TP fragment, is generated by PCR amplification. The amplified fragment of Tgf2TP was then used together with the pEGFP-N1 plasmid to construct the pEGFP-Tgf2TP plasmid, which was then used together with the SV40-NLS fragment to construct the pEGFP-TP-SV40NLS plasmid, using the primer sequences shown in Table 3 below:

TABLE 3 primers used for amplification of fragments of interest

1.2.2.2 PCR amplification

As the upstream primer and the downstream primer are respectively added with enzyme cutting recognition site bases, in order to ensure the following double enzyme cutting efficiency, protective bases (italic marks) are respectively added at the 5 'end of the upstream primer and the 3' end of the downstream primer. PCR amplification was performed using pGEX-4T-1-Tgf2TP plasmid as a template, and the reaction system and procedure are as follows:

TABLE 4 transposase DNA fragment PCR amplification System

1.2.2.3 electrophoretic detection

A1.2% agarose gel was prepared according to the following formula: 5

TABLE 5 agarose gel formulations

And after the detection electrophoresis is finished, photographing and storing.

1.2.2.4 agarose gel recovery

1) The concentration of the recovered rubber is 1.5%, and the formula is shown in Table 5; 2) observing the migration position of the DNA band under an ultraviolet lamp after electrophoresis is finished, cutting off a gel block containing the target band, and adding the gel block into a sterilized centrifugal tube; 3) recovering the target band according to the steps of the specification in the agarose gel recovery kit; 4) after completion of the recovery, the recovered product was electrophoretically detected and the DNA concentration was measured.

1.2.3 construction of pMD19-Tgf2TP recombinant plasmid

1.2.3.1 ligation and transformation

The recovered Tgf2TP fragment was ligated with pMD19-T vector by the following procedure (Table 6).

TABLE 6 reaction System, procedure

The ligation products were inoculated into 100. mu.L of competent cells, mixed and placed in a refrigerator at 4 ℃ for 30 minutes, transferred to a water bath at 42 ℃ for heat shock for 90s, and then quickly placed on ice for 5 min. 890. mu.L of LB liquid medium was added and incubated at 37 ℃ for 60 min. And centrifuging the obtained bacterial liquid at a low speed, then removing about 750 mu L of supernatant, coating the residual bacterial liquid on the surface of a selective LB (Amp or Kan) solid culture medium, and performing inverted incubation culture at 37 ℃.

1.2.3.2 identification of PCR amplification products

Dipping a small amount of single colonies for shake bacteria amplification culture, taking the single colonies as a template for PCR amplification, and selecting bacterial liquid conforming to a target band after electrophoresis detection for sequencing by workers. And comparing the sequencing results to known sequences.

1.2.3.3 double digestion and plasmid recombination

The positive clone obtained in the experiment is named as pMD19-Tgf2TP, and a bacterial colony liquid with correct sequencing is selected and inoculated in an LB liquid (including Amp resistance) culture medium for amplification culture. After the plasmid was extracted and the concentration of the plasmid was measured, double digestion of the plasmid was performed, and the digestion system is shown in Table 7. The pMD19-Tgf2TP was double-digested with XhoI/BamHI restriction enzymes to obtain the objective DNA fragment having cohesive ends.

Meanwhile, the empty-load plasmid pEGFP-N1 stored in a laboratory is transformed into escherichia coli DH5 α competent cells for amplification and plasmid extraction, and then the plasmid is detected through electrophoresis and the concentration is measured.

TABLE 7 reaction systems and procedures

And (3) carrying out electrophoresis detection after enzyme digestion, carrying out gel cutting recovery treatment on the enzyme digestion product if the size of an electrophoresis strip accords with that of a target strip, and storing the enzyme digestion product at-20 ℃ for later use after marking.

1.2.4 construction of pEGFP-Tgf2TP recombinant plasmid

The Tgf2TP product recovered from the double-restriction gel cutting and the pEGFP-N1 cleavage product were combined in the following order of 5: 1, the ligation reaction was carried out. The attachment system and procedure are as follows: 8

TABLE 8 reaction System, procedure

And then, transforming, plating and culturing the connection product, carrying out bacterium selection, bacterium liquid culture and PCR amplification on the next day, selecting bacterium liquid with the consistent size of the band after electrophoresis detection, and sending the bacterium liquid to a sequencing department for sequencing. And if the sequencing is correct, the pEGFP-Tgf2TP recombinant plasmid is constructed, then bacterium liquid with correct sequencing is subjected to shake bacterium amplification, and then the plasmid is extracted and stored for later use.

1.2.5 construction of pEGFP-TP-SV40NLS recombinant plasmid

Because the SV40NLS fragment is very short, the sequence is directly sent to a synthetic part for synthesis according to a primer synthesis method, the viscous tail end of a BamHI enzyme cutting site is added at the upstream-5 'end of the sequence, and the viscous tail end of an AgeI enzyme cutting site is added at the downstream-3'. Then, the two complementary single strands are annealed by an annealing process in a PCR instrument to generate double-stranded DNA. The specific method comprises dissolving the primers with annealing buffer solution, mixing the primers to be annealed at equal molar number, heating to 95 deg.C for 5min, and slowly cooling to room temperature (below 30 deg.C, and the cooling process is controlled at about 1 hr). The annealed product can be stored in a refrigerator at 4 ℃.

The pEGFP-Tgf2TP recombinant plasmid obtained above was subjected to double digestion using BamHI and AgeI rapid endonucleases in the following system: 9.

TABLE 9 reaction systems and procedures

After the cleavage, the detection was carried out by electrophoresis using the plasmid pEGFP-Tgf2TP as a control. And after electrophoresis detection, if the size of the electrophoresis strip is in accordance with the size of the electrophoresis strip, performing rubber tapping recovery treatment on the enzyme digestion product, marking the recovered product, and storing the product in a storage box at the temperature of-20 ℃ for later use.

The product recovered by double digestion of pEGFP-Tgf2TP recovered from the above tapping was mixed with SV40NLS having a cohesive end in the following order of 1: 5, and then the ligation reaction was carried out in a 10. mu.L system. The attachment system and procedure are as follows: 10

TABLE 10 reaction systems and procedures

And then transforming, plating and overnight culturing the connection product, carrying out bacteria selection, bacteria shaking and PCR reaction on the next day, selecting bacteria liquid according with the size of a strip after electrophoresis detection, and sending the bacteria liquid to a sequencing department for sequencing. And if the sequencing is correct, the pEGFP-TP-SV40NLS recombinant plasmid is constructed, then the bacterial liquid with the correct sequencing is subjected to amplification culture, and then the plasmid is extracted and stored for later use.

1.2.6 transposase mRNA preparation

1.2.6.1 Single enzyme digestion

The recombinant plasmids pEGFP-Tgf2TP and pEGFP-TP-SV40NLS were subjected to single-enzyme digestion to obtain linearized recombinant plasmids, see Table 11.

TABLE 11 reaction System and procedure

And (3) carrying out electrophoresis detection after enzyme digestion is finished, then carrying out gel cutting recovery on the DNA product, wherein the recovery concentration is kept to be more than 200 ng/mu L.

1.2.6.2 in vitro transcription

The linearized pEGFP-Tgf2TP and pEGFP-TP-SV40NLS transposase plasmids recovered in the previous step were used as templates for in vitro transcription, and the reaction system and procedure are shown in the following table: 12

TABLE 12 in vitro transcription reaction System and procedure

In the table, RNA Sin @ is Recombinan RNase @ Ribonucleae Inhibitor.

1) After the reaction, 30. mu.L of RNA free DEPC H was added to the tube ₂Mixing O and 2.5 mu L of 4M LiCl solution uniformly, and standing at-20 ℃ for 1 h; 2) centrifuging the sample in a centrifuge at 14000rpm at 4 ℃ for 15min, and discarding the supernatant; 3) adding 1000 μ L70% ethanol water solution (ready for use), and centrifuging for 15 min; 4) carefully sucking the supernatant and abandoning, and standing for 6min at room temperature; 5) add 30. mu. LDEPC H ₂O and 0.3 μ LRNA inhibitor; 6) detecting the product by electrophoresis; 7) after the detection is finished, the in vitro transcription product is placed in a low-temperature storage box at the temperature of-80 ℃ for storage so as to avoid degradation.

1.2.6.3 microinjection and progeny culture

The evening before microinjection, separating male and female zebra fish suitable for production by a baffle plate, removing the baffle plate before microinjection, uniformly placing fertilized eggs in a groove of a microinjection plate after the fertilized eggs lay eggs, sucking surrounding water but not drying, respectively mixing pTgf2-EF1 α -eGFP donor plasmid and pEGFP-Tgf2TP transposase mRNA, and pTgf2-EF1 α -eGFP donor plasmid and pEGFP-TP-SV40NLS transposase mRNA in a ratio of 1:1.5, and then adding phenol red indicator and driving into the microinjection needle.

After the microinjection needle is installed, the button on the microinjection instrument is adjusted to adjust the optimum microinjection pressure and the volume of the drug. After the optimal state is adjusted, the fertilized eggs of zebra fish which have developed to 1-2 stages are injected (about 2 nL). The injection can keep the action as fast and soft as possible, and the mechanical damage to the fertilized egg can be reduced. After the injection is finished, the fertilized eggs are gently placed in a culture dish filled with aeration water at the temperature of about 28 ℃ for incubation, and the aeration water is changed in time and dead eggs are sucked away every 4 hours. The development of the embryos was observed and recorded periodically. After the young fish is taken out of the film and the mouth is opened, the paramecium is added into the film for feeding, and after the young fish can be fed, the paramecium is fed with the artemia cysts.

1.2.7 cell experiments (all experiments were carried out in clean bench)

1.2.7.1 cell culture

The cell subculture steps are as follows:

1) the old culture solution in the HeLa cell culture bottle to be passaged is sucked out and discarded. 2) 4-6ml of PBS was added to the flask to rinse the cell surface, and the wash solution was discarded. This step was repeated twice. 3) 1-2ml of trypsin was added to the cell culture flask, taking care that the cell surface was not directly washed. The digestion step may be in CO ₂In an incubator. After digestion, the cells are moved to a microscope for observation, and if the cell morphology is observed to be deformed and shrunk, the pancreatin is quickly sucked out, or new culture medium is directly added to stop the digestion process. 4) Then, about 6ml of pre-prepared DMEM was added to the digested cell culture flask, and the bottom of the flask was blown as much as possible to prepare a cell suspension, with gentle action. 5) Inoculating HeLa cell suspension into new culture bottles according to required proportion, adding 6ml DMEM into each new culture bottle, slightly shaking the culture bottles to uniformly distribute cells, and placing the culture bottles in CO ₂Incubator (5% CO) ₂37 ℃ C.).

1.2.7.2 transient transfection of cells

1) Adherent HeLa cells to be passaged were made into cell suspensions the day before the transient transfection experiment (note: the medium used at this point should be free of diabodies) and then added to 24-well plates, 500. mu.L of cells to be transfected were added to each well, and then the 24-well plates were placed in CO ₂Culturing in an incubator; 2) calculating the dilution volume according to the plasmid concentration on the next day, and uniformly mixing and diluting the plasmid DNA in 50 mu L of an Opti-MEM serum-free culture medium; 3) then 2. mu.L of transfection reagent is taken and diluted in 50. mu.L of LOpti-MEM, and is kept stand for 5 min; 4) subsequently, the samples from 2) and 3) were mixed with CO ₂Placing in an incubator 2And 0 min. 5) Finally, the transfection solution is evenly added into a 24-hole plate paved on the previous day in a dropwise manner in CO ₂Culturing in an incubator; 6) After 24h, the 24-well plate was inverted under an inverted fluorescence microscope for observation, diluted at a ratio of 1:8, and DMEM was added for late stage counting.

1.2.7.3 DAPI staining

1) After observing the transfected cells under a microscope, removing DMEM in a 24-well plate, and adding PBS into the well for rinsing for about 2-3 times; 2) adding 4% paraformaldehyde into each transfection well for fixation, and standing for 20min in a dark place; 3) adding 500 mu L of DAPI staining solution into each transfection hole to perform cell nucleus staining on the transfected HeLa cells, and standing for 15min in a dark place; 4) after the DAPI liquid is removed, rinsing with PBS for 3 times; 5) the expression of green fluorescence was observed under a Leica inverted fluorescence microscope and stored by photographing.

1.3 results of the experiment

1.3.1 construction of pEGFP-TP plasmid

The results of electrophoresis after PCR amplification using the template plasmid pGE-4T-1-Tgf2TP are shown in FIG. 1.

Since the electrophoretic bands are single and correspond to the length of the fragment of Tgf2TP, we recovered the target band by tapping. After the steps of connection transformation, plate coating, bacteria selection, bacteria shaking, PCR detection and the like, electrophoresis detection is carried out, and the result is shown in figure 2:

and selecting bacterial liquid which meets the size of the target band, and sending the bacterial liquid to a worker for sequencing. The sequencing result is consistent with our target fragment, and has XhoI cutting site at the 5 'end and BamHI cutting site at the 3' end, and the sequence between the two cutting sites is the coding sequence of gene with GenBank accession number HM 146132.

And (3) shaking the bacterial liquid with the correct sequencing and extracting a plasmid, which is named as PMD-19T-Tgf2 TP. After the plasmid extraction was completed, concentration detection was performed, and then the double digestion of PMD-19T-Tgf2TP and pEGFP-N1 plasmids, respectively, was performed as shown in FIG. 3.

Recovering Tgf2TP and pEGFP-N1 vector fragments after double digestion respectively, and then mixing the target fragments with a vector 3: 1, the ligation reaction was carried out. And then, quickly transforming the ligation product, selecting a single colony for bacteria shaking after plating and culturing, and then carrying out PCR (polymerase chain reaction), wherein the electrophoresis detection result is shown in figure 4.

Because the obtained bands are single and regular, one tube of the bacterial liquid is selected and sent to a bio-company for sequencing. The sequencing result proves that the fragment digested by the Tgf2TP and the fragment digested by the pEGFP-N1 vector are successfully connected, namely the pEGFP-Tgf2TP plasmid is successfully constructed, as shown in FIG. 5.

And continuously culturing and amplifying the bacterial liquid which is sequenced successfully, extracting pEGFP-TP plasmid, detecting the concentration, and storing in a refrigerator at minus 20 ℃ and minus 80 ℃ for later use.

1.3.2 construction of pEGFP-TP-SV40NLS plasmid

Since we directly synthesized and annealed to form a double-stranded SV40NLS fragment with BamHI cleavage site at the upstream-5 'end and AgeI cleavage site at the downstream-3', the plasmid pEGFP-Tgf2TP needs to be subjected to BamHI and AgeI double cleavage, and the cleavage results are shown in FIG. 6.

And (3) performing gel tapping recovery on the fragments which are subjected to double enzyme digestion and conform to the size of a target band (because the enzyme digestion sites of BamHI and AgeI are very close, an electrophoresis band cannot be displayed after double enzyme digestion is finished), connecting the fragments with an SV40NLS fragment, quickly converting the SV40NLS fragment which is positioned at the downstream of a Tgf2TP coding sequence into an escherichia coli DH5 α competent cell, selecting a single bacterial colony for bacteria shaking after plate coating culture, performing PCR (polymerase chain reaction), and performing electrophoresis detection, wherein the electrophoresis detection result is shown in a figure 7.

And selecting corresponding bacterial liquid which meets the size of a target band, sending the corresponding bacterial liquid to a sequencing department for sequencing, confirming that the fragment cut by pEGFP-Tgf2TP is successfully connected with the SV40NLS fragment with the viscous tail end through a sequencing result, and then carrying out amplification culture on the bacterial liquid to extract pEGFP-TP-SV40NLS plasmid (the structure is shown in figure 8).

1.3.3 Zebra Fish microinjection results

We used pTgf2-EF1 α -eGFP transposon plasmid as donor plasmid, and pEGFP-Tgf2TP and pEGFP-TP-SV40NLS plasmids as auxiliary plasmids, and then the auxiliary plasmid was linearized and reverse transcribed into mRNA, and microinjected into zebrafish eggs together with the donor plasmid and phenol red.

The results show that the fluorescence expression of the positive control group and the experimental group, which are injected with helper plasmids pEGFP-Tgf2TP and pEGFP-TP-SV40NLS, is greatly increased compared with the signal intensity of a transposon plasmid pTgf 2-1 α -eGFP only, the fluorescence expression observed at 12h after microinjection is unevenly distributed, the fluorescence expression of the positive control group and the experimental group, which is also increased compared with the negative control group, the fluorescence expression observed at 24h after microinjection is generally expressed, the fluorescence expression of the positive control group and the experimental group, which is also observed at 12h after the microinjection, is generally expressed in whole body, the fluorescence expression of the positive control group and the experimental group, which is also increased compared with the negative control group, the fluorescence expression of the positive control group and the experimental group, which is generally expressed in whole body, is generally concentrated at body, the fluorescence expression of the positive control group and the experimental group, which is also greatly increased compared with the negative control group, the fluorescence expression of the positive control group and the experimental group, which are also significantly expressed in whole body, and the positive control group, which is also significantly increased in total expression of Tef 2, and the transposition enzyme (Tef-Tf) is significantly increased in whole), which is significantly improved compared with the positive control group, which is significantly expressed in whole).

1.3.4 integration efficiency statistics

After the zebrafish were injected to the membrane and observed for fluorescence, we kept it until it matured according to the breeding method described previously. Then cutting the tail fin of the adult zebra fish, and extracting DNA of the adult zebra fish injected with the live zebra fish by using a marine organism genome DNA extraction kit. Then, the genome of the live injected zebra fish is detected by PCR to determine whether the GFP gene is carried. The detection results are shown in FIG. 12.

The detection of the zebra fish surviving microinjection shows that the integration efficiency of GFP in the negative control group is about 8.3 percent; in our positive control group, the integration efficiency can reach 48.1% after the co-injection of pEGFP-Tgf2TP transposase auxiliary plasmid mRNA; the integration efficiency of the experimental group injected with pEGFP-TP-SV40NLS transposase helper plasmid mRNA is improved by 60.8% compared with the positive control group (Table 13). It was found by analysis that a transposase helper plasmid containing SV40NLS can help to increase the integration efficiency of Tgf2 transposon.

TABLE 13 statistics of GFP positivity after microinjection

1.3.5 cell transfection

We transfected the constructed pEGFP-Tgf2TP and pEGFP-TP-SV40NLS plasmids, respectively, and the control pEGFP-N1 plasmid into HeLa cells, observed the expression of green fluorescent protein 24h after transfection, and then stained the cell nucleus with DAPI dye. The green fluorescence expression was observed as in FIG. 13.

Since all passive and active transport into and out of the nucleus occurs through NPC, the structure and function of NPC appears to be conserved in all eukaryotes, suggesting its central role in eukaryotic cell function. NPC has the function of a porous molecular sieve, and molecules with the molecular weight of less than 40-45kDa can freely diffuse into and out of cell nucleus. Whereas proteins larger than 45kDa require NLS to aid their transport into the nucleus. Since pEGFP-N1 has a molecular weight of approximately 27kD, it is free to diffuse through NPC into the nucleus, and GFP is uniformly expressed in the nucleus and cytoplasm, as shown in FIG. 13. The molecular weight of pEGFP-Tgf2TP and pEGFP-TP-SV40NLS plasmids constructed by the method is more than 45kDa, so that the NLS sequence carried by transposase needs to be used for helping the plasmid to enter the nucleus.

1.3.6 transfection efficiency assay

For quantitative analysis, we performed routine analysis on 200-: n, indicating cells that express fluorescence only in the nucleus, N > C, indicating cells that express more nuclear fluorescence than cytoplasm; n ═ C, meaning cells with a uniform distribution of fluorescent expression in the nucleus and cytoplasm; n < C, indicating that the fluorescence intensity in the cytoplasm exceeds that of cells expressed in the nucleus. As shown in fig. 14.

After fluorescence analysis, we found that when the pEGFP-N1 plasmid of the transfection control group enters HeLa cells, green fluorescence is uniformly expressed in cytoplasm and nucleus. After transfection of the pEGFP-Tgf2TP transposase plasmid into HeLa cells, complete expression of GFP in the nucleus was observed in 40.05% of the cells; GFP can be expressed both in the cytoplasm and the nucleus in 56.97% of cells, and more GFP is expressed in the nucleus than in the cytoplasm in 2.98% of cells. When the plasmid of pEGFP-TP-SV40NLS transposase is transfected into HeLa cells, the increase of the cell number of GFP which is completely expressed in cell nucleus can be observed, and the cell number reaches 51.63%; while the number of homogeneous expression in cytoplasm and nucleus decreased to only 45.60%, the GFP expression in nucleus was 2.77% more than in cytoplasm (see Table 14).

TABLE 14 statistics of GFP positivity after microinjection

1.4 sequence description to which the invention relates

The sequence of Tgf2 transposase (Tgf2TP) used herein was derived from goldfish

>pEGFP-Tgf2TP

The coding sequence of Tgf2TP was 2016bp, as indicated by the underlined section, and the sequence followed by EGFP was not a stop codon for the transposase sequence. Wherein the first box shows the recognition site for Xho I (followed by the start codon), the shaded sequence is the recognition site for BamH I (the last codon upstream of which is the last codon of the coding sequence), and the second box is the recognition site for Age I.

>pEGFP-TP-SV40NLS

Tgf2TP (coding sequence 2016bp, designated as the first underlined sequence: SEQ ID NO.1), SV40NLS coding sequence as the second underlined sequence (designated: SEQ ID NO.2), the first box sequence Xho I recognition site, shaded BamH I recognition site, the second box sequence Age Age I recognition site.

The protein sequence corresponding to the Tgf2 transposase is as follows:

MFIGPLEVTSCHIYYHNAQHLDLEIREIITVNQWKKMEEVCDSSAAASSTVQNQPQD QEHPWPYLREFFSFSGVNEDSFKMKCVLCLPLNKEISAFKSSPSNLRKHIERMHPNYLKNY SKLTAQKRKIGTSTHASSSKQLKVDSVFPVKHVSPVTVNKAILRYVIQGLHPFSTVDLPSFK ELISTLQPGISVITRPTLRSKIAEAALIMKQKVTAAMSEVECNFIGITAHWINPGSLERHSAA LACKRLMGSHTFEVLASAMNDIHSEYEIRDKVVCTTTDSGSNFLKAFRVFGVENNDIETE ARRCESDDTDSEGCGEGSDGVEFQDASRVLDQDDGFEFQLPKHQKCACHLLNLVSSVDA QKALSNEHYKKLYRSVFGKCQALWNKSSRSALAAEAVESESRLQLLRPNQTRWNSTFMA VDGILQIYKEAGEGALQNICTSLEVPMFNPAEMLFLTEWANTMRPVAKVLDILQAETNTQ LGWLLPSVHQLSLKLQRLHHSLRYCDPLVDALQQGIQTRFKHMFEDPEIIAAAILLPKFRTS WTNDETIIKRGMDYIRVHLEPLDHKKELANSSSDDEDFFASLKPTTHEASKELDEYLACVS DTRESLLTFPAICSLSIKTNTPLPASAACERLFSTAGLLFSPKRARLDTNNFENQLLLKLNLRFYNFE (named as SEQ ID NO. 3).

The protein sequence corresponding to SV40NLS is as follows:

CTPPKKKRKV (named as SEQ ID NO. 4).

1.5 discussion

In recent years, research and application of transposon-mediated insertional mutagenesis in various fields of human and mammals have been extensively conductedThe application is as follows. The kit is used as a simple and efficient tool carrier in genetic biological analysis, and comprises the following components: has important effects in the research of molecular genetic level, cell level, animal individual level, transgenic animal model establishment, gene therapy and the like [9]. It is known that the transposable element functions by requiring a helper plasmid, i.e., a transposase plasmid, to provide mRNA or a plasmid constructed on the basis of transposase to assist the transposase in functioning. The difficulty in this case is to construct suitable helper plasmids which are capable of transcription in organisms, since in most organisms relatively few genes are available which provide the promoter necessary for the helper plasmid ^[10]. The goldfish Tgf2 transposon discovered by the laboratory has the activity of autonomous transposition, is a promising vertebrate transgenic vector, and can be developed and utilized as a transgenic research.

In our experimental results, the SV40NLS sequence was increased at the C-terminus of the Tgf2 transposase protein, and the integration efficiency was indeed increased both in nuclear entry efficiency and after microinjection.

(one) conclusion

The Tgf2 transposon discovered in goldfish by the laboratory team still maintains the activity of autonomous transposition, can encode transposase, and can exert the function of autonomous transposition. This experiment has conducted some studies on how to improve the transposition function of a Tgf2 transposable element, and it is expected that it will provide a basis for better functioning of a Tgf2 transposable element. The preliminary results obtained are as follows:

(1) tgf2 transposon donor plasmid pTgf2-EF1 α -eGFP with green fluorescent protein GFP gene, transposase plasmids pEGFP-Tgf2TPmRNA and pEGFP-TP-SV40NLS mRNA are respectively subjected to coinjective microinjection and introduced into fertilized eggs of zebra fish, the green fluorescent protein of the reporter gene is detected by observation at different periods of embryonic development after in vitro culture, and the positive rate is detected by sequencing, the result shows that the positive rate of the transposase plasmid containing SV40NLS through microinjection is improved by 12.7 percent relative to a control group, which shows that SV40NLS can help to improve the integration efficiency and the transposition efficiency of Tgf2 transposable elements.

(2) The transposase plasmids pEGFP-Tgf2TP and pEGFP-TP-SV40NLS are transfected into HeLa cells respectively, and the observation of green fluorescence in the transfected HeLa cells shows that the green fluorescence of the recombinant pEGFP-Tgf2TP-SV40NLS transposase protein is mostly concentrated in nuclear distribution, and compared with the cells of a control group which is transiently transfected with pEGFP-Tgf2TP transposase, the nuclear entry efficiency is improved by 11.58%. The SV40NLS can help to improve the nuclear entry efficiency and further improve the transposition efficiency.

Looking at

Tgf2 transposon as a DNA transposon with autonomous transposition activity can code transposase, and has wide research prospect. Since transposases having high activity are known to have NLS sequences, the development and utilization of a Tgf2 transposase is a problem to be solved at present. Therefore, the main research directions in the future are:

(1) the transfection ratio of transposon and transposase in different species or cells can affect the integration efficiency of the foreign gene. Therefore, it is suggested that the optimum transfection ratio of the Tgf2 transposon and the transposase thereof can be optimized, and the optimum ratio can be screened out, which can help us to search the optimum microinjection ratio, thereby improving the transposition efficiency.

(2) Since mRNA may be toxic during microinjection and affect transposition efficiency, we considered to construct a plasmid containing both a Tgf2 transposon and a transposase. So as to make the best proposal for the development and the utilization of a Tgf2 transposition system and provide a cornerstone for the wide application of the transposition element.

(3) Combining the above experimental results, we can use the Tgf2 transposable element as the basis of the insertional mutagenesis study in zebrafish, and use the transposon to search the hypoxia-resistant mechanism of megalobrama amblycephala in the later study.

The above embodiments are only used for further illustration of the present invention, and are not intended to limit the scope of the present invention, and all equivalent changes made based on the concept of the present invention and obvious modifications of various technical solutions of the present invention fall within the scope of the present invention.

Sequence listing

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Val Phe Pro Val Lys His Val Ser Pro Val Thr Val Asn Lys Ala Ile

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tcaaacctaa ggaagcatat tgagagaatg cacccaaatt acctcaaaaa ctactctaaa 360

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Claims

1. A recombinant transposase comprising a protein sequence comprising a Tgf2 protein sequence and an NLS protein sequence.

2. The recombinant transposase of claim 1, wherein the coding sequence of the Tgf2 protein sequence has GenBank accession number: HM 146132.

3. The recombinant transposase of claim 1 wherein the NLS protein sequence is an SV40NLS protein sequence.

4. The recombinant transposase of claim 1 wherein the Tgf2 protein sequence is upstream of the NLS protein sequence.

5. The recombinant transposase of claim 1, wherein the sequence of Tgf2 protein is as shown in SEQ ID No. 1; the NLS protein sequence is shown in SEQ ID NO. 2.

6. A recombinant transposase gene capable of encoding the recombinant transposase of any one of claims 1-4.

7. The recombinant transposase gene as claimed in claim 6, wherein a coding sequence of gf2 protein in the recombinant transposase gene is represented by SEQ ID No. 3; the coding sequence of the NLS protein sequence is shown as SEQ ID NO. 4.

8. A recombinant expression vector comprising the recombinant transposase gene as claimed in claim 6 or 7.

9. Use of the recombinant transposase of any one of claims 1-5 or the recombinant transposase gene of claim 6 or 7 or the recombinant expression vector of claim 8 for increasing the efficiency of fish transposition.

10. The use according to claim 9, wherein the fish is selected from the group consisting of megalobrama amblycephala, goldfish, and zebrafish.