CN109402153B

CN109402153B - Construction of protein label vector and expression and detection method thereof

Info

Publication number: CN109402153B
Application number: CN201811418693.0A
Authority: CN
Inventors: 陈冬生; 陶小倩; 孙铭钟; 吴华勤
Original assignee: Anhui Normal University
Current assignee: Anhui Normal University
Priority date: 2018-11-26
Filing date: 2018-11-26
Publication date: 2021-11-05
Anticipated expiration: 2038-11-26
Also published as: CN109402153A

Abstract

The invention discloses a construction of a protein label vector and an expression and detection method thereof, relating to the technical field of biomarkers, and the invention takes a nos gene with high specificity expression of drosophila ovary germ cells as an example to develop a protein labeling technology of cell endogenesis, namely a protein label technology based on end exon labeling; firstly, constructing a transgenic vector in which the last exon of the nos gene is embedded with a GFP tag sequence by means of a molecular cloning technology; secondly, the drosophila carrying the transgenic vector is prepared by means of the mature transgenic technology of the drosophila, and whether the fluorescence of the expression product GFP of the nos gene in the ovarian cells is expressed or not is detected, so that the technology provides convenience for the research of the expression mode of the drosophila gene of the model organism and provides reference for the research of the expression and the positioning of the mammalian gene.

Description

Construction of protein label vector and expression and detection method thereof

Technical Field

The invention relates to the technical field of biomarkers, in particular to a construction method of a protein label vector and an expression and detection method thereof.

Background

The RNA splicing process is a process of excising a non-coding region called an intron from a pre-mRNA molecule and splicing coding regions called exons in a gene to form a mature mRNA. It is a very important biological process in eukaryotic gene expression, and through RNA splicing, many functional mRNAs with coding information can be generated, which is important for the development and evolution of organisms. The RNA spliceosome is responsible for the RNA splicing process, specifically, the RNA is cut at the junction of exon and intron of the initial transcription product of mRNA (prefrosor mRNA, pre-mRNA for short), then the exons are sequentially connected, and the intron is cut off to form a complete mRNA. Studies have shown that splice sites of pre-mRNA in eukaryotic cells have certain sequence conservation, and bases at the 5 'end (donor site) and 3' end (acceptor site) of an intron are almost GT and AG, so the GT-AG rule is called. Most of the existing researches focus on the aspects of sequence recognition mechanism of splicing sites, in-vitro reconstruction of splicing, splicing regulation and the like.

The GFP protein is called Green Fluorescent Protein (GFP) and is found in jellyfish in 1962 by Nomura et al, encoded by the GFP gene. The protein can emit green fluorescence under the excitation of blue wavelength light, and the characteristic is that GFP protein is widely used as a label protein in the field of life science. The GFP mediated fusion protein labeling technology is a recombinant DNA technology based on a reporter gene, which is started at the end of the 20 th century, and can be applied to detection of in vivo gene expression level, intracellular in-situ tracing and positioning of protein, purification of target protein, enhancement of solubility and stability of recombinant protein and the like. The gene nos (referred to as nos for short) is very conserved and widely exists in a plurality of species such as nematodes, silkworms, zebrafish, magains, mice and humans. nos plays an important role in regulating and controlling ontogenesis, maintenance of germ cells, migration and determination of primordial germ cells and the like. In Drosophila, the nos gene is highly expressed in early embryonic cells, primordial germ cells, mature reproductive organs, ovary and testis.

Based on the research on mRNA splicing principle, GFP protein labeling technology, molecular cloning technology and mature Drosophila transgenic technology, the inventor finds that the expression mode of Drosophila gene is that the fusion sequence of GFP and target gene coding sequence is first set behind the target gene promoter to constitute Drosophila transgenic vector; then obtaining the transgenic fruit fly by means of microinjection so as to trace the expression mode and subcellular localization of the target protein. There are the following problems: first, there is uncertainty in the effective promoter length of the target gene. The promoter may be ineffective if designed to be too short, does not express GFP fusion protein, and is difficult to design for too long amplification. Secondly, after the transgenic vector constructed by genetic engineering means is integrated into the drosophila genome, the expression of the fusion gene is the imitation of the endogenous target gene, and is slightly different from the in-vivo in-situ expression. Thirdly, the coding sequence of the target gene requires extraction of total mRNA from specific tissues, followed by reverse transcription to obtain cDNA, and finally by gene amplification. Compared with the technology developed by the patent, the whole process is time-consuming and labor-consuming, and the cost is increased.

Disclosure of Invention

In view of the above, the present invention is directed to a protein-tagged vector, and its expression and detection method, for overcoming all or part of the above-mentioned deficiencies in the prior art.

Based on the above purpose, the invention provides a method for constructing a protein tag vector, which comprises the following steps:

(1) amplifying a 3'UTR-AS sequence of the drosophila nos gene to obtain nos3' UTR-AS, wherein the base sequence is SEQ ID NO. 1;

(2) constructing a pattern B-nosP-3' UTR-AS transition vector;

(3) amplifying the upstream 4550bp sequence of the act5c gene ATG to obtain an alkali sequence before amplification as SEQ ID NO. 2;

(4) constructing a pattB-act5cP-3' UTR-AS transition vector;

(5) amplification of the sequences of nosE3D-I3-E4C and GFP, wherein the base sequence of GFP is SEQ ID NO. 3;

(6) amplification of the sequence of nosE 3D-I3-E4C-GFP;

(7) construction of pattB-act5cP-nosMLExon-GFP vector, wherein the base sequence of nosMLExon is SEQ ID NO. 4.

In some alternative embodiments, the 3' UTR-AS sequence is the drosophila nos gene terminal exon 3' untranslated region sequence 3' UTR, and its downstream 100bp additional sequence AS.

In some alternative embodiments, the step (2) requires carrying restriction sites of restriction enzymes Sbf1 or Age1, Kpn1, Asc1, Not1 and Xba1 in nos3' UTR-AS during construction, and Kpn1, Asc1 and Not1 are pre-buried cleavage sites.

In some alternative embodiments, the step (3) is amplification using a gene of a wild type drosophila as a template.

In some alternative embodiments, said step (4) is amplification by recombinant PCR techniques.

In some alternative embodiments, the step (6) adopts a bridging method for amplification, namely two pairs of primers are designed, firstly, the nosE3D-I3-E4C and GFP are respectively amplified, and then, the nosE3D-I3-E4C and GFP are used as templates, and the overlapping part between the two templates is used for bridging amplification and is inserted into the vector once.

In some alternative embodiments, during the amplification by the bridging method, restriction enzyme sites can be introduced into the nosE3D-I3-E4C and GFP respectively, and the restriction enzyme sites can be inserted into transition vectors respectively for amplification.

In some alternative embodiments, the amplification of the sequence nos E3D-I3-E4C in step (5) has no TAG stop codon, and the GFP sequence is amplified by introducing a TAG stop codon at its 3' end.

As can be seen from the above, the invention constructs the transgenic vector of the 'end exon-GFP fusion sequence', and by means of mature drosophila transgenic technology, the mRNA of a target gene (nos gene) can be fluorescently labeled; under the condition of not influencing the in-situ expression of the target gene, the RNA splicing process of the target gene is spontaneously embedded into an mRNA product, so that the expression level and the expression mode of the target gene can be truly reflected, the subcellular localization of the target protein can be reflected, the time and the labor are saved, and the effect of achieving multiple purposes is achieved. The technology provides convenience for the expression pattern research of the pattern organism drosophila gene and provides reference for the research of expression and positioning of the mammalian gene.

The invention also aims to provide a protein tag vector expression method and a detection method, wherein the protein tag vector gene prepared by the method is injected into the eggs of the drosophila melanogaster in a micro-injection manner, and then red-eye transgenic drosophila melanogaster is selected to establish a transgenic drosophila melanogaster strain.

A protein label carrier detection method is characterized in that prepared ovaries and spermary of transgenic drosophila melanogaster are taken for immunofluorescence staining, and GFP expression detection is carried out.

As can be seen from the above, the transgenic recombinant vector of the protein tag marked by the terminal exon is constructed, the protein tag is fused into the splicing model of the chimeric gene, and GFP fluorescence localization confirms that the splicing model of the chimeric exon-intron-exon-protein tag (GFP) is added into the RNA splicing process in an organism.

Drawings

FIG. 1 is a schematic diagram showing the sequence of 3' UTR-AS of a nos gene amplified by PCR in an embodiment of the present invention, M, 2000 DNA marker, 1, 980bp target band under ultraviolet light;

FIG. 2 is a schematic diagram showing the PCR identification of the recombinant plasmid pattB-nosP-3' UTR-AS of the embodiment of the present invention, M, 2000 DNA marker, 1-14, band of 980bp under UV light, + AS positive control, -AS negative control;

FIG. 3 is a schematic diagram showing the double enzyme digestion identification of Sbf1 and Xba1 of recombinant plasmid pattB-nosP-3' UTR-AS according to the embodiment of the present invention, wherein M and 15000 DNA marker, 1 and 2 are the enzyme digestion bands of number 1 and number 2 positive transformants respectively;

FIG. 4 is a schematic diagram showing the amplification of the act5cP4.55k sequence of example of the present invention, M, 15000 DNA marker, 1, 4.55kb target band under UV light;

FIG. 5 is a schematic diagram of the PCR identification of the recombinant plasmid pattB-act5cP-3' UTR-AS according to the embodiment of the present invention, wherein M, 2000 DNA marker, + is a positive control, 1-11 is a band of 420bp under UV light, and-is a negative control;

FIG. 6 is a schematic diagram of the double restriction enzyme identification of Sbf1 and Kpn1 of the recombinant plasmid pattB-act5cP-3' UTR-AS of the embodiment of the present invention, wherein M and 15000 DNA markers, 1 and 2 are the restriction enzyme bands of number 6 and number 7 positive transformants, respectively;

FIG. 7 is a schematic diagram of the sequence of GFP and nosE3D-I3-E4C amplified by PCR in the example of the present invention, M, 2000 DNA marker, target bands of about 714bp and 394bp under UV light in 1, 2;

FIG. 8 is a schematic diagram showing the sequence of PCR amplification of nosE3D-I3-E4C-GFP of the example of the present invention, M, 2000 DNA marker, 1 is a target band of about 1108bp under UV light;

FIG. 9 is a schematic diagram of the PCR identification of the recombinant plasmid ATB-act5cP4.5k-ATG-nos Exon L-GFP-nos3' UTR of the present invention, M, 2000 DNA marker, + is the positive control, 1-10 is the band of about 1108bp under UV light, and-is the negative control;

FIG. 10 is a schematic diagram of the double restriction enzyme identification of Kpn1 and Not1 of recombinant plasmid pattB-act5 cP-nonsMLExon-GFP in the example of the present invention, wherein M and 15000 DNA markers, 1 and 2 are the restriction enzyme bands of the

number

1 and 3 positive transformants, respectively;

FIG. 11 is a diagram showing the construction of the pattern B-act5cP-nosMLExon-GFP transgenic vector according to the embodiment of the present invention;

FIG. 12 is a diagram of a pattern B-act5 cP-nonsMLExon-GFP Drosophila transgenic vector according to the present invention;

FIG. 13 is a graph showing Immunohistochemical (IHC) staining patterns of pattB-act5 cP-nonsMLExon-GFP transgenic fruit flies and control fruit fly (Oregon), with GFP antibody staining in the square circles and cell nuclei staining in the circular circles, according to the example of the present invention.

Detailed Description

It should be noted that the following biotechnological terms are referred to in the examples of the present invention:

(1) end exon labeling: first, a gene (here, a nos gene, which is called a target gene) "sequence of a part downstream of the penultimate Exon (here, the 3 rd Exon of the nos gene, Exon 3)", "the last Intron (here, the 3 rd Intron of the nos gene, Intron 3)", "the last Exon (here, the 4 th Exon of the nos gene, Exon 4) and a sequence 100bp downstream thereof" in a genome is amplified and cloned, and is called a target sequence. Then, a TAG sequence (e.g., GFP) is inserted in front of a stop codon (e.g., TAG) following the internal coding sequence of the terminal exon of the target sequence, thereby obtaining an altered target sequence. Finally, the modified target sequence is carried by a transgenic vector and integrated into a genome, and the modified target sequence can participate in RNA splicing and protein translation processes of target genes in cells. Finally, the label sequence and the coding protein of the target gene sequence form a fusion protein, and the target protein expression and the subcellular localization thereof can be traced.

(2) Exon (Exon): part of eukaryotic disruption gene is coding sequence. It is preserved after splicing and can be expressed as a protein during protein biosynthesis.

(3) Intron (Intron): a part of a eukaryotic disrupted gene can be transcribed, but is spliced out during mRNA processing. Here the nos gene contains 4 exons and 3 introns.

(4) Protein labeling: the tag protein is connected with the target protein coding sequence by using a DNA in vitro recombination technology and is used as a transgenic sequence to transform cells. The tag protein will be co-expressed with the target protein to form a fusion protein. The presence of the tag protein facilitates detection, tracking, purification, etc. of the target protein.

(5) The "pattB-" type drosophila transgenic vector: a transgenic carrier for the site-specific insertion of fruit fly. Can carry exogenous gene to be inserted into the genome of Drosophila at fixed point.

(6) act5 cP: the promoter of act5c gene. Comprises a 4550bp (base pair) sequence fragment upstream of the start codon ATG of the act5c gene. The act5c gene is housekeeping gene and is expressed in eukaryotic cell in broad spectrum.

(7) Drosophila nanos gene: abbreviated as nos. The nos gene encodes the nos protein. Drosophila Nos gene can be expressed in early embryonic cells and germ line cells of ovary, and Nos protein expressed in ovary is important for differentiation and development of germ cells.

(8) NosMLExon-GFP: abbreviated as "nos-Modified Last Exon-GFP". The expression refers to the modification of the end exon of the nos gene with a GFP tag sequence. Specifically, the modified sequence comprises the following components in sequence from upstream to downstream: the Sequence Downstream of the penultimate Exon (Exon 3) of nos gene (Exon 3-Downstroke Sequence, E3D), the final Intron (Intron 3, I3), the final Exon (Exon 4) Coding Sequence (Exon 4-Coding Sequence, E4C), the GFP tag fusion Sequence, the final Exon 3' untranslated Region Sequence (3' Un-Translational Region,3' UTR), and the 100bp (base pair) Additional Sequence (AS). Is totally named AS nosE3D-I3-E4C-GFP-3' UTR-AS.

(9) GFP protein: green Fluorescent Protein (GFP), which is found in jellyfish by Nomura et al and encoded by the GFP gene. The protein can emit green fluorescence under the excitation of blue wavelength light. Can be used as a label protein and applied to detection of in vivo gene expression, subcellular localization of protein and the like.

(10) Drosophila transgenic vector and transgenic Drosophila: DNA molecules for transformation of Drosophila. Most of the DNA molecules are covalent, closed and circular plasmid DNA and can be integrated into the nuclear genome DNA of early embryonic cells of drosophila. The fruit fly carrying the transgenic vector is named as transgenic fruit fly, and the fruit fly uses the red eye rescuing gene carried on the transgenic vector as a marker for screening and transforming the fruit fly.

DNA Marker: standard reference fragment of DNA length.

DH 5. alpha.: a recipient bacterium capable of taking up exogenous DNA is a mutagenized strain.

pattern B-nosP: a vector which can be inserted into the chromosome of Drosophila at a certain site.

PET28 a: a prokaryotic expression vector.

CIAP: an acid phosphatase is provided. The phosphate group at the 5' end of the vector DNA fragment may be removed.

Amp +: resistance to ampicillin.

And (3) PCR: i.e., polymerase chain reaction. DNA is denatured at 95 ℃ in vitro to become single-stranded, a primer and the single-stranded DNA are combined according to the principle of base complementary pairing at low temperature (usually about 60 ℃), the temperature is adjusted to the optimal reaction temperature (about 72 ℃) of DNA polymerase, and the DNA polymerase synthesizes a complementary strand along the direction from phosphoric acid to pentose (5 '-3').

Restriction enzymes: an endonuclease which hydrolyzes double-stranded DNA at a specific nucleotide sequence.

DNA ligase: a nickase enzyme on a closed DNA strand catalyzes the formation of a phosphodiester bond between 5'-PO4 on one DNA strand and the 3' -OH on the other DNA strand by the energy provided by ATP or NADP hydrolysis. However, these two strands must be paired with the same complementary strand (except for T4DNA ligase), and two adjacent DNA strands must be catalyzed by DNA ligase to form phosphodiester bonds.

Construction of the Gene expression vector: the process of combining the gene of interest with the carrier is actually a process of recombining DNAs from different sources. If a plasmid is used as the carrier, the plasmid is first cut with a restriction enzyme to create a nick in the plasmid and expose the cohesive ends. Then, the same restriction enzyme is used for cutting the target gene, so that the same cohesive end is generated (the flat end can be cut by partial restriction enzyme, and the same effect is achieved). Inserting the cut target gene segment into the cut of plasmid, base complementary pairing combination, making two cohesive ends anastomotic together to form hydrogen bond between bases, adding proper amount of DNA ligase to catalyze two DNA chains to form phosphodiester bond, and connecting adjacent DNA to form a recombinant DNA molecule. This recombinant DNA molecule is also called recombinant plasmid.

And (3) IHC: namely, immunohistochemistry, also called immunocytochemistry, refers to that fluorescent or chromophoric chemical substances are combined on an antibody, and the presence of a target antigen in a cell or tissue is detected by utilizing the specific combination reaction between an antigen and the antibody in the immunology theory, so that the method can be used for measuring the expression amount of a protein and carrying out protein localization.

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to specific embodiments and drawings.

An object of an embodiment of the present invention is to provide a method for constructing a protein tag vector, which is characterized by comprising the following steps:

(1) amplifying a 3'UTR-AS sequence of the drosophila nos gene to obtain a nos3' UTR-AS, wherein the base sequence is SEQ ID NO. 1;

(2) constructing a pattern B-nosP-3' UTR-AS transition vector;

(4) constructing a pattB-act5cP-3' UTR-AS transition vector;

(6) amplification of the sequence of nosE 3D-I3-E4C-GFP;

The method comprises the following specific steps:

1. amplification of Drosophila nos Gene 3' UTR-AS sequence

A pair of specific primers (namely H1 and H2) are designed and synthesized, and cutting sites Sbf1, Kpn1, Asc1, Not1 and Xba1 of several restriction enzymes which do Not appear in the 3' UTR-AS sequence of the nos gene are respectively introduced into H1 and H2, wherein Kpn1, Asc1 and Not1 are cutting sites which are embedded in advance for later use, and Sbf1 can be replaced by Age 1.

Italicized letters indicate the Kpn1, Asc1, Not1 cleavage sites, respectively;

adding the two primers into a PCR reaction system, wherein the PCR amplification conditions are as follows: after the cycle of denaturation at 95 ℃ for 3mim, denaturation at 95 ℃ for 15s, annealing at 55 ℃ for 15s, and extension at 72 ℃ for 30s is completed for 35 cycles, extension at 72 ℃ is continued for 5min, and a 980bp target fragment with an Sbf1, Kpn1, Asc1, Not1 and Xba1 enzyme cutting site at the end is obtained (as shown in FIG. 1).

2. Construction of Pattern B-nosP-3' UTR-AS transition vector

After the PCR product of 980bp length was purified and the pattB-nosP vector was digested with restriction enzymes Sbf1 and Xba 137 ℃ for 1 hour, the pattB-nosP vector was digested for 1 hour, 1. mu.L of CIAP was added to remove phosphorus to prevent self-ligation of the vector, the mixture was incubated at 37 ℃ for 30min, and the nos3' UTR-AS sequence was inserted into the pattB-nosP plasmid using T4 ligase (16 ℃ C., overnight ligation). The ligation product was introduced into E.coli DH5 alpha competent cells, positive clones were selected from a solid medium containing ampicillin resistance (Amp +), amplified by PCR (see FIG. 2), and the recombinant plasmid was identified by double digestion with Sbf1 and Xba1 (see FIG. 3).

As can be seen from the figure: FIG. 2 shows a 980bp fragment, and FIG. 3 shows that the PattB-nosP-3' UTR-AS plasmid was digested simultaneously with Sbf1 and Xba1 to obtain a small fragment of about 980bp and a large fragment of about 10000 bp. The 980bp exogenous fragment is proved to be inserted into the plasmid, PCR and enzyme cutting results show that a pattB-nosP-3' UTR-AS plasmid vector is successfully prepared, a bacteria liquid sample of the positive clone is sent to a biological company for sequencing, and the sequencing result is compared with a target sequence. As a result, it was found that the nos3' UTR-AS 980 bp-long sequence inserted into the pattB-nosP vector completely agreed with the target sequence.

3. amplification of the sequence of 4550bp upstream of ATG of act5c gene (act5cP4.55k)

A pair of specific primers (namely H3 and H4) are designed, restriction sites Sbf1 and Kpn1 which do not appear in the act5cP4.55k are respectively introduced into H3 and H4, and the act5cP4.55k sequence is amplified by PCR by taking the drosophila genome as a template. The PCR amplification conditions were as follows: performing 95 ℃ denaturation for 3mim, performing 95 ℃ denaturation for 15s, annealing at 55 ℃ for 15s, and extending at 72 ℃ for 20s, and performing 35 cycles and then continuing extension at 72 ℃ for 5min to obtain the target fragment (shown in figure 3).

As a result of PCR, FIG. 4 shows a 4.55kb band, and it was confirmed that the target fragment of act5cP4.55 kb was obtained in accordance with the theory.

4. Construction of Pattern-act 5cP-3' UTR-AS transition vector

After purifying the PCR product of 4.55kb length amplified above and digesting the pattern B-nosP-3' UTR-AS vector with restriction enzymes Sbf1 and Kpn 137 ℃ for 1 hour, 1. mu.L of CIAP was added after digesting the pattern B-nosP-3' UTR-AS vector for 1 hour, incubating at 37 ℃ for 30min, and inserting the act5cP4.55k fragment into the pattern B-nosP-3' UTR-AS plasmid with T4 ligase (16 ℃ C., overnight ligation). The ligation product was introduced into E.coli DH5 alpha competent cells, positive clones were selected from Amp + -containing solid medium, amplified by PCR (results shown in FIG. 5), and the recombinant plasmid was identified by double digestion using Sbf1 and Kpn1 (results shown in FIG. 6).

As can be seen from the figure: FIG. 5 shows a fragment of about 420bp (a sequence of 420bp within the act5cP4.55k fragment), and FIG. 6 shows that the pattern B-act5cP-3' UTR-AS plasmid is subjected to double digestion with Sbf1 and Kpn1 to obtain a small fragment of 4.55kb and a large fragment of about 11000 bp. Proved that a 4.55kb fragment is inserted into the plasmid, PCR and enzyme cutting results show that a pattB-act5cP-3' UTR-AS plasmid vector is successfully prepared, a bacterial liquid sample of the positive clone is sent to a biological company for sequencing, and the sequencing result is compared with a target sequence. As a result, it was found that the sequence of act5cP4.55k inserted into the pattB-nosP-3' UTR-AS vector was correct. When the act5cP4.55k and the nosE3D-I3-E4C-GFP were inserted, instead of inserting the act5cP4.55k first, the present invention can be also achieved by inserting the nosE3D-I3-E4C-GFP first and then inserting the act5cP4.55k.

5. Amplification of the sequences of nos E3D-I3-E4C and GFP

Two pairs of specific bridging primers (namely H5, H6, H7 and H8) are designed and synthesized, and nosE3D-I3-E4C and restriction enzyme cutting sites Kpn1 and Not1 of restriction enzymes which do Not appear in GFP are respectively introduced into H5 and H8, and an initiation codon ATG and pre-buried Asc1 are introduced after H5 Kpn 1.

Bold letters indicate the initiation codon ATG;

the stop codon TAG is indicated in bold italics.

The genome and the pLenti6.3-IRES-GFP plasmid are respectively taken as templates, two pairs of primers are respectively added into the two PCR reaction systems, and the sequences of the nosE3D-I3-E4C and GFP are amplified. The PCR amplification conditions were as follows: after the cycle of denaturation at 95 ℃ for 3mim, denaturation at 95 ℃ for 15s, annealing at 55 ℃ for 15s, and extension at 72 ℃ for 25s, and after 35 cycles, extension at 72 ℃ is continued for 5min, 394bp NosExon L fragment with Kpn1 enzyme cutting site and initiation codon ATG at the end and 714bp GFP sequence with termination codon TAG and enzyme cutting site Not1 at the end are obtained respectively (see FIG. 7).

When the nosE3D-I3-E4C-GFP fragment is amplified by the bridging method PCR, restriction enzyme sites are respectively introduced into the nosE3D-I3-E4C and the GFP and are respectively inserted into transition vectors, so that the invention can be realized.

6. Amplification of the sequence of nosE3D-I3-E4C-GFP

The amplified nos E3D-I3-E4C and GFP sequences are respectively taken as templates, and bridging primers H5 and H8 are added to the overlapping parts of the two fragments to amplify the nos E3D-I3-E4C-GFP sequences. The PCR amplification conditions were as follows: 3mim denaturation at 95 ℃ enters a cycle, denaturation at 95 ℃ is carried out for 15s, annealing at 55 ℃ is carried out for 15s, extension at 72 ℃ is carried out for 35s, and after 35 cycles, extension is carried out for 5min at 72 ℃, so as to obtain 1108bp of the objective sequence of nosE3D-I3-E4C-GFP (shown in figure 8).

7. Construction of Pattern B-act5cP-nosMLExon-GFP vector

The amplified nosE3D-I3-E4C-GFP sequence and the constructed pattB-act5cP-3' UTR-AS plasmid are digested for 1h by restriction enzymes Kpn1 and Not 137 ℃, after the pattB-act5cP-3' UTR-AS plasmid is digested for 1h, 1 mu L of CIAP is added, incubation is carried out for 30min at 37 ℃, and the nosE3D-I3-E4C-GFP sequence is inserted into the pattB-act5cP-3' UTR-AS plasmid by T4 ligase (16 ℃, overnight ligation). The ligation product was introduced into E.coli DH5 alpha competent cells, positive clones were selected from Amp + -containing solid medium, and the recombinant plasmid was identified by double digestion with PCR (see FIG. 9) and Kpn1, Not1 (see FIG. 10).

As can be seen from the figure: FIG. 9 shows a 1108bp fragment, and FIG. 10 shows that the pattern B-act5 cP-nonsMLExon-GFP plasmid is subjected to Kpn1 and Not1 double digestion to obtain a small fragment of about 1108bp and a large fragment of more than 10000bp, which proves that the drosophila expression vector pattern B-act5 cP-nonsMLExon-GFP is successfully prepared. And (3) sending the bacteria liquid sample of the positive clone to a biological company for sequencing, and comparing a sequencing result with a target sequence to obtain a correct sequence.

8. Transgenic drosophila preparation and GFP gene expression detection

The Drosophila transgenic vector constructed as described above (pat B-act5 cP-nosMLon-GFP) was microinjected into Drosophila eggs following published procedures (see Bischof J, Maeda RK, Hediger M, Karch F, & Basler K. an optimized transgenic system for Drosophila using germ-line-specific phiC phi C31 [ J ]. Proc Natl Acad Sci USA,2007,104 (9): 3312-Occupied) and red-eye transgenic Drosophila was selected to establish transgenic Drosophila lines, see FIGS. 11 and 12.

According to the experimental methods in the documents 1 and 2 (document 1: old winter, wang shuang, wang sword, periazalea, majoram, & sunglong luck & preparation and identification of drosophila hsp83 polyclonal antibody, China histochemistry and cytochemistry journal, 2016,25(5),454 + 459. document 2: Zhuxiang, wang shuang, wang sword, Cao Zhijie, Liuxurong, and Chen winter student. study on half-life of the reproductive stem cells of three drosophila testis, laser biology reports, 2016,25(2),156 + 160), ovaries and sperms of transgenic drosophila are respectively taken for immunofluorescence staining, and the expression of GFP is detected as shown in FIG. 13. The immunohistochemical staining result of the transgenic drosophila melanogaster shows that the GFP expression mode is a nos gene expression mode, and no GFP expression exists in a control group, so that the nos MLExon-GFP in the vector constructed by the invention participates in the splicing process of RNA in drosophila melanogaster, and the nos gene expression is reflected to a certain extent.

Those of ordinary skill in the art will understand that: the discussion of any embodiment above is meant to be exemplary only, and is not intended to intimate that the scope of the disclosure, including the claims, is limited to these examples; within the idea of the invention, also features in the above embodiments or in different embodiments may be combined, steps may be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.

The embodiments of the invention are intended to embrace all such alternatives, modifications and variances that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, substitutions, improvements and the like that may be made without departing from the spirit and principles of the invention are intended to be included within the scope of the invention.

SEQUENCE LISTING

<110> university of teacher's university in Anhui

<120> construction of protein tag vector and expression and detection method thereof

<130> 2018-11-22

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 980

<212> DNA

<213> Drosophila melanogaster (Drosophila melanogaster)

<400> 1

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ggtctgcaac tggtcgcctt catttcgtaa aatgttcgct tgcggccgaa aaatttcgat 420

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actttttgat agataagcga agagtatttt tattacatgt atcgcaagta ttcatttcaa 660

cacacatatc tatatatata tatatatata tatatatata tatatatata tatgttatat 720

atttattcaa ttttgtttac cattgatcaa tttttcacac atgaaacaac cgccagcatt 780

atataatttt tttatttttt taaaaaatgt gtacacatat tctgaaaatg aaaaattcaa 840

tggctcgagt gccaaataaa gaaatggtta caatttaagg aaacaaatgt ccttcttgcg 900

tttgaaacaa ctaatccttt tcgccctcgc ggcgtttctc gaaaagggcc aggaagatgc 960

catcggtaag atcactgtcc 980

<210> 2

<211> 4550

<212> DNA

<213> Drosophila melanogaster (Drosophila melanogaster)

<400> 2

cctgcaggcc gaagccattg gcccggggaa atttctgcga gttttttcat cgccttgcca 60

aaaaaaaaaa aaaacaaaaa acaaacgatc gagcaacgaa cttgagagaa gcaaatagag 120

ccaaaaaaaa ggttatgatt ttgttccaaa gaatgttcca tagaattcta tattctaaaa 180

acacaaatga tacttctaaa aaaaaatcat attcataaat gactctttcc atgaatggca 240

tcaactctga atcaaatctt tgcagatgca cctacttctc atttccactg tcacatcatt 300

tttccagatc tcgctgcctg ttatgtggcc cacaaaccaa gacacgtttt atggccatta 360

aagctggctg atcgtcgcca aacaccaaat acataatgaa tatgtacaca ttcgagaaag 420

aagcgatcaa agaagcgtct tcgggcggag taggagaatg cggaggagaa ggagaacgag 480

ctgatctagt atctctccac aatccaatgc caactgacca actggccata ttcggagcaa 540

tttgaagcca atttccatcg cctggcgatc gctccattct tggctatatg tttttcaccg 600

ttacccgggg ccattttcaa agactcgtcg gcaagataag attgtgtcac tcgctgtctc 660

tcttcatttg tcgaagaatg ctgaggaatt tcgcgatgac gtcggcgagt attttgaaga 720

atgagaataa tttgtattta tacgaaaatc agttagtgga attttctaca aaaacatgtt 780

atctatagat aattttgttg caaaatatgt tgactatgac aaagaactgg aattaagtgg 840

catattttgc attgctttcc gataatagtt gtatttttat aaagtaaagc gtttgtaaaa 900

taaatacctt taatgtattc tcattttctt atgtatttat aatggcaatg atgatactga 960

tgatatttta agatgatgcc agaccaaaag gcttgaattt ctgcgtcttt tgccgaacgc 1020

agtgcatgtg caattgttgt tttttggaat attcaatttt cggactgtcc gctttgattt 1080

cagtttcttg gcttattcaa aaagcaaagt aaagccaaaa aagcgagatg gcaataccaa 1140

atgcggcaaa acggtagtgg aaggaaaggg gtgcggggca gcggaaggaa gggtggggcg 1200

gggcgtggcg gggtctgtgg ctgggcgcga cgtcaccgac gttggagcca ctcctttgac 1260

catgtgtgcg tgtgtgtatt attcgtgtct cgccactcgc cggttgtttt tttcttttta 1320

tgctgcgctc tctctagcgc catctcgctt acgcatgctc aacgcaccgc atgttgccgt 1380

ttccttttat gcgtcatttt ggctcgaaat aggcaattat ttaaacaaag attagtcaac 1440

gaaaacgcta aaataaataa gtctacaata tggttactta ttgccatgtg tgtgcagcca 1500

acgatagcaa caaaagcaac aacacaggtg gctttccctc tttcactttt tgtttgcaag 1560

ccgcgtgcga gcaagacggc acgaccggca aacgcaatta cgctgacaaa gagcagacga 1620

agttttggcg aaaaacatca aggcgcctga tacgaatgca tttgcaataa caattgcgat 1680

atttaatatt gtttatgaag ctgtttgact tcaaaacaca caaaaaaaaa aataaaacaa 1740

attatttgaa agagaattag gaatcggacg cttatcgtta gggtaacaac aagaaatgct 1800

tactgagtca cagcctctgg aaaactgccg caagccagag agagagagaa aaagagggag 1860

agcagcttag accgcatgtg cttgtgtgtg aggcgtctct ctcttcgtct ctgttgcgca 1920

aacgcataga ctgcactgaa aaaatcgatt acctattttt tatgaatgaa tatttgcact 1980

attactattc aaaactatta agatagcaat cacattcaat agccaaatac tataccacct 2040

gagcgatgca acgaaatgat caatttgagc aaaaatgctg catatttagg acggcatcat 2100

tatagaaatg cttcttgctg tgtacttttc tctcgtctgg cagctgtttc gccgttattg 2160

ttaaaaccgg cttaagttag gtgtgttttc tacgactagt gaatgcccta ctagaagatg 2220

tgtgttgcac aaaatgtccc tggaataacc aatttgaagt gcagatagca gtaaacgtaa 2280

gctaatatga atattattta actgtaatgt tttaatatcg ctggacatta ctaataaacc 2340

cactataaac acatgtacat atgtatgttt tggcatacaa tgagtagttg gggaaaaaat 2400

gtgtaaaagc accgtgacca tcacagcata aagataacca gctgaagtat cgaatatgag 2460

taacccccaa attgaatcac atgccgcaac tgataggacc catggaagta cactcttcat 2520

ggcgatatac aagacacaca caagcacgaa cacccagttg cggaggaaat tctccgtaaa 2580

tgaaaaccca atcggcgaac aattcatacc catatatggt aaaagttttg aacgcgactt 2640

gagagcggag agcattgcgg ctgataaggt tttagcgcta agcgggcttt ataaaacggg 2700

ctgcgggacc agttttcata tcactaccgt ttgagttctt gtgctgtgtg gatactcctc 2760

ccgacacaaa gccgctccat cagccagcag tcgtctaatc cagagacacc aaaccgaaag 2820

acttaattta tatttattta attaatttta ataaaacaca ccaaatgtaa gtagctttcc 2880

ccttcccaac aacaaaacac catcgaacca ctcccaccaa gaaaaagcaa taatcgagaa 2940

aagccgcgga aaatgtgtga ttttttttgt aaacaaaaca tttttttatg tgccagtgct 3000

gaaagtgatc aaaaaatact agccacgagc taaagagtta ttgtattgac caaaactcca 3060

aaaataccca agtttggccc taaattgtca atcaaaatac caataggtcg aaagacatca 3120

aaattaacaa aaccagggtt tcaaatacca taactcaaga atcaggatta caactgcaga 3180

tttcaggata tatacataca aattatacga aattataaaa accaaagcaa ttcaatagcc 3240

ccaactcaaa tgttaggatc taatatagtg tttaaagcca agctcgctga tgtgggcgtg 3300

tcacgatttc acccaaagat atgccaaatt acgaattgca aatcaattcg ccaacacttt 3360

ctttttttcc cacgccctaa aacaccagat catccataaa tgtacataca tacagtatat 3420

gcatattata atctgtaaaa ctagatcagg ttcttgaaaa tagtgacgta ggcagccgtt 3480

ttggctgaag cagaaatttt tgccggtttt tcaaagttgt agttgcaaaa atggagaaaa 3540

ccttcgagca ttcgttcata tacacacact cacgcgcaaa ataacgagag agagtgtatg 3600

tgtgtgtgag agagcgaaag ccagacgacg gtttgctttt cgcctcgaaa catgaccata 3660

tatggtcaca aaacttggcc gccgcaattc aacacaccag cgctctcctt cgcacccata 3720

gcgaccatgg cgcggagcga gcgagatggc gagagcgagc gacgcctatg gcgacgtcga 3780

cgcaggcagc gattgaaaaa cgcagttaac tggcattcaa cattcaccag ccactttcag 3840

tcggtttatt ccagtcattc ctttcaaacc gtgcggtcgc ttagctcagc ctcgccactt 3900

gcgtttacag tagttttcac gccttgaatt tgttaaatcg aacaaaaagg taaagtttaa 3960

ctagctttga aaagtttcgt ggctcttaat tgttaaattt tctagagtgc gtttagtgtt 4020

tttttttttt tttattttgt aatgttaatt tcgggttcca attcgagttt taggcagccg 4080

ccattttaag ggcgcataca cacaggcaac tgtgctctct ttgcggcttt cttttgcacc 4140

ggcattcgtt aagtgtcgtc tagaagcttc tcccctccct tttcggcata ttcgtattgt 4200

ggttttaatt tttcggggcg gggccttcta ttttgtaact gttcttttaa tttcttatta 4260

caattcgatc gcaagtgaaa atcagttttc aatcggaaaa gtattttttt atgaaatttt 4320

ttttgtccaa gattaaaatt ttgtactaaa aaaacgtaca ttgcattgag tgatttttaa 4380

ttgtacacga aaaacaagtt agtttgttat gacaattgta ctttggtaga ccagcgcagt 4440

ccaaggaaac cacgcaaatt ctcagttttt tttttgccat ttctacatta ccaaataagg 4500

taaccaaaaa ctaatgggaa atccgcattc tttccattgc agcttacaaa 4550

<210> 3

<211> 714

<212> DNA

<213> Drosophila melanogaster (Drosophila melanogaster)

<400> 3

gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 60

gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc 120

aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 180

gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag 240

cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc 300

aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 360

aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag 420

ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc 480

atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac 540

cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac 600

ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg 660

ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caag 714

<210> 4

<211> 421

<212> DNA

<213> Drosophila melanogaster (Drosophila melanogaster)

<400> 4

ggtaccatgg gcgcgccaga atagtaaagt aacaataata acaacaacaa caaggtgtac 60

aagcgttaca acagcaaggc caaagaggtg agtggatctg atccaggatt cgcagaatta 120

aaacttgcaa aatatatttg ttatcttgtt tctccaacag atcagccgcc actgcgtctt 180

ttgtgagaat aacaacgaac cagaggcggt tatcaatagc cactcagtgc gagataactt 240

taaccgagtg ctgtgcccca aactacgcac ctacgtgtgc cccatctgcg gggcatctgg 300

ggactcggcg cacacgatta agtactgccc caagaagccg atcatcacca tggaggatgc 360

gatcaaggcg gaatcgttcc gcctagccaa gagcagttac tacaagcaac agatgaaggt 420

t 421

Claims

1. A method for constructing a protein tag vector pattB-act5cP-nosMLExon-GFP is characterized by comprising the following steps:

(1) fruit flynosAmplifying a gene 3'UTR-AS sequence to obtain nos3' UTR-AS, wherein the base sequence is SEQ ID NO. 1;

(2) construction of pattB-nosP-3' UTR-AS transition vector: purifying the PCR product obtained in the step (1), carrying out enzyme digestion reaction on the purified PCR product and a pattB-nosP plasmid by using restriction enzyme, and then inserting a nos3' UTR-AS sequence into the pattB-nosP plasmid by using T4 ligase;

(3) amplifying the sequence of the upstream 4550bp of the act5c gene ATG to obtain the base sequence SEQ ID NO.2 after amplification;

(4) construction of pattB-act5cP-3' UTR-AS transition vector: carrying out enzyme digestion reaction on the PCR product obtained in the step (3) and a pattB-nosP-3'UTR-AS vector by using a restriction endonuclease after purification, and then inserting an act5cP4.55k fragment into a pattB-nosP-3' UTR-AS plasmid by using T4 ligase;

(6) amplification of the sequence of nosE 3D-I3-E4C-GFP;

(7) construction of protein-tagged vector pattB-act5cP-nosMLExon-GFP vector: the sequence of nosE3D-I3-E4C-GFP was inserted into the pattB-act5cP-3' UTR-AS plasmid, in which the base sequence of nosMLExon is SEQ ID NO. 4.

2. The method of claim 1, wherein the 3' UTR-AS sequence is drosophila melanogasternos3'UTR of a 3' untranslated region sequence of a terminal exon of the gene and an additional sequence AS of 100bp downstream of the sequence.

3. The construction method according to claim 1, wherein in the construction process of step (2), restriction sites for restriction enzymes Sbf1, Kpn1, Asc1, Not1 and Xba1 are required to be carried on nos3' UTR-AS, and Kpn1, Asc1 and Not1 are pre-buried cleavage sites, wherein Sbf1 can be replaced by Age 1.

4. The method according to claim 1, wherein the step (3) is carried out by amplifying the gene of the wild type Drosophila melanogaster as a template.

5. The method of claim 1, wherein the step (6) is performed by a bridge-approach amplification method, in which two pairs of primers are designed, and the nos E3D-I3-E4C and GFP are amplified separately, and then the nos E3D-I3-E4C and GFP are used as templates, and the templates are inserted into the vector at a time by a bridge-approach amplification method using the overlapping portion between the two templates.

6. The method of claim 1, wherein the step (6) comprises amplifying by a bridge-bypass method, introducing restriction sites into the nos E3D-I3-E4C and GFP, and inserting a transition vector into each of them.

7. The method of constructing a GFP product according to claim 1, wherein the amplification of the sequence nos E3D-I3-E4C in step (5) is free of a TAG stop codon, and the GFP sequence is amplified by introducing a TAG stop codon at the 3' end thereof.

8. A protein tag vector expression method, characterized in that, the coding gene of the protein tag vector prepared by any one of the methods of claims 1-7 is injected into the eggs of Drosophila melanogaster by micro-injection, then red-eye transgenic Drosophila melanogaster is selected, and transgenic Drosophila melanogaster strains are established.

9. A protein tag vector detection method, characterized in that the ovary and the testis of the transgenic fruit fly established in claim 8 are taken, immunofluorescent staining is carried out, and expression detection of GFP is carried out.