CN108486113B - 2A lytic peptide-based multigene element for realizing equivalent expression of two genes, expression vector and application - Google Patents

Abstract

The invention relates to a 2A lytic peptide-based multigene element for realizing equivalent expression of two genes, an expression vector and application, wherein the gene element consists of Ck and 2A lytic peptide; the nucleotide sequence of the 2A cleavage peptide is shown as SEQ ID NO. 38; the nucleotide sequence of Ck is shown in SEQ ID NO.39, and the vector containing the element can equivalently recombine and express genes at two ends of 2A, and has important significance for realizing multi-gene expression of silkworms.

Description

2A lytic peptide-based multigene element for realizing equivalent expression of two genes, expression vector and application

Technical Field

The invention belongs to the technical field of biology, and relates to a 2A lytic peptide-based multigene element for realizing equivalent expression of two genes, an expression vector containing the element and application.

Background

The efficient recombinant expression of multiple genes in silkworm bodies can promote the basic and application research of silkworms, such as the research of the interrelation among multiple genes, the recombinant expression of the gene of a multi-subunit protein complex, the visual research of the movement of a target gene in vivo by expressing fluorescent protein simultaneously, the cultivation of silkworm varieties capable of resisting the bombyx mori cytoplasmic polyhedrosis virus and the nuclear polyhedrosis virus, and the like. Owing to the establishment of transgenic operation system of silkworm in 2000, the simultaneous expression of several genes in silkworm body has been realized mainly through genetic hybridization. Firstly, a plurality of transgenic silkworm strains expressing a target gene singly must be established, then hybridization is carried out, and finally silkworm individuals expressing a plurality of exogenous genes simultaneously are screened from filial generations. For example, in 2006, Takahiro Adachi et al successfully expressed bombyx mori prolyl hydroxylase β -subunit and human collagen simultaneously in the posterior silk gland of bombyx mori; in 2009, Masashizuka et al expressed both the light chain gene and the heavy chain gene of a murine monoclonal antibody, and expressed the complete murine monoclonal antibody on the silkworm cocoon shell; in 2015, Minoru Tada et al also expressed anti-CD20 monoclonal antibody recombinantly in the middle silk gland of Bombyx mori using Gal4/UAS system in a two-round hybridization manner. The method not only takes a long time, but also has a complex hybridization process.

The 2A lytic peptide is an oligopeptide found in 1991 in foot and mouth disease virus, which is present between two proteins (2A, 2B), and usually has 19-22 amino acids. When the ribosome reaches the last two amino acids glycine and proline at the C-terminal of 2A, the downstream gene cannot be translated due to the change of the internal structure of the ribosome amide center, at this time, the upstream nascent peptide is hydrolyzed to release the ribosome, and then the downstream gene is translated by the ribosome, so that the equivalent expression of the two genes can be theoretically realized. Currently, 2A has successfully co-expressed multiple combinations of genes in multiple cell lines, species (mouse, zebrafish, pig, drosophila and sheep species) for the treatment of cancer and other diseases, or for genetic improvement of plants and new species of animals with specific functions or resistance. Therefore, in the previous studies, we constructed a bombyx mori silk gland 2A polygene expression system using P2A for porcine enterovirus, but this system could not express the DsRed gene and EGFP gene upstream and downstream of 2A in equal amounts by recombination.

Kozak sequence (Kozak consensus sequence) is a nucleic acid sequence located after the 5 'end cap structure of eukaryotic mRNA, usually GCCACCAUGG, which can bind to a translation initiation factor to mediate translation initiation of mRNA containing the 5' cap structure. In 1981, when the KOZAK of female scientists studied the influence of site-directed mutagenesis of bases around the initiation codon AUG in eukaryotes on the transcription and translation of target genes, it was first concluded that in eukaryotes, the two terminal sequences of the initiation codon are: the highest transcription and translation efficiency was observed at-G/N-C/N-C/N-ANNAUGG-GCCACCAUGG, GCCAUGAUGG, and particularly, the-3-position A and the + 4-position G were important for the translation efficiency. Currently, many researchers have also reported that Kozak sequence can affect the expression level of gene. For example, in 2014, the expression level of WNT16 gene was improved by 3.7 times by optimizing the regulatory sequence (GCACCC) upstream of the gene to Kozak sequence (GCCACC), and the expression level of GATA4 gene was severely reduced by changing the G mutation at position-6 of the Kozak sequence upstream of GATA4 gene to C.

However, the Kozak sequence is used for optimizing a 2A polygene expression system in the silk gland of the silkworm, so that the gene located at both ends of 2A can be equivalently recombined and expressed, and no report is found.

Disclosure of Invention

In view of the above, it is an object of the present invention to provide a 2A cleavage peptide-based genetic element that achieves equivalent expression of two genes; the second object of the present invention is to provide a multigene expression vector containing the gene element; the third purpose of the invention is to provide the application of the gene element.

In order to achieve the above purpose, the invention provides the following technical scheme:

1. a 2A lytic peptide-based genetic element that effects equal expression of two genes, the genetic element consisting of Ck and 2A lytic peptide; the nucleotide sequence of Ck is shown as SEQ ID NO. 39.

Preferably, the gene element is formed by connecting a P2A sequence of the pig enterovirus optimized by the GSG joint and Sk, and the P2A sequence of the pig enterovirus optimized by the GSG joint is shown as SEQ ID NO. 38; the Sk nucleotide sequence is shown as SEQ ID NO. 40.

2. A multigene expression vector containing said genetic elements.

Preferably, the expression vector is shown as SEQ ID NO. 36.

3. The use of said genetic elements for achieving equal expression of multiple genes.

The invention has the beneficial effects that: the invention discloses a 2A lytic peptide-based gene element for realizing equivalent expression of two genes, which optimizes a 2A polygene expression system in a silk gland of a silkworm by utilizing a Kozak sequence, so that the gene expression system can equivalently recombine and express genes positioned at two ends of 2A, and provides technical support for recombining and expressing a plurality of genes in the silk gland and silk of the silkworm.

Drawings

In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1 shows that Ck enhances the expression level of downstream genes by increasing the translation efficiency in Sf9 cells (A: the structure diagram of transient expression vector, k is a sequence designed according to kozak rule; B: the fluorescence result after transfection of Sf9 cells with transient expression vector; scale: 400 um; C: RT-PCR analysis of DsRed gene transcription in Sf9 cells; D: the expression of RFP in Sf9 cells after transfection; D: the relative content of RFP in Sf9 cells after transfection).

FIG. 2 shows Ck-enhanced expression level of downstream EGFP gene of BmCPV2A (30) in BmE cells (A: transient expression vector structure diagram; Sk is kozak sequence at N-terminal of sericin 1 gene of endogenous gene of Bombyx mori; B: RT-PCR detection of transcription level of fusion gene after transfection of BmE cells by transient expression vector; C: fluorescence result after transfection of Sf9 cells by transient expression vector with scale of 400 um; D: expression conditions of RFP and EGFP in BmE cells after transfection; E and F are relative expression quantity analysis of RFP and EGFP in BmE cells after transfection, respectively.

FIG. 3 shows that Ck mediates efficient expression of DsRed and EGFP genes in the middle silk gland of transgenic silkworm pR-CkG (A: transgenic expression vector, red arrow is RT-PCR primer, B: fluorescence result in middle silk gland of transgenic silkworm pR-CkG, scale is 2mm, C and D are RT-PCR detection of transcription of fusion gene DsRed-P2A-CkEGFP in the middle silk gland of normal silkworm and pR-CkG transgenic silkworm, respectively, E: detection of recombinant proteins in the middle silk gland of transgenic silkworm pR-CkG, total protein concentration in lane pR-CkG-1 is 1/2 in pR-CkG-2, 1/4 in pR-CkG-3, red and green pentagons indicate successfully disrupted RFP and EGFP proteins, respectively, and content analysis of recombinant proteins RFP and EGFP in the middle silk gland of F transgenic silkworm pR-CkG).

FIG. 4 shows the expression of DsRed gene and EGFP gene in transgenic silkworm pCkG-CkR (A: transgenic expression vector; B: screening of positive transgenic silkworm moth; C and D: RT-PCR detection of the transcription of fusion gene CkG-CkR in the middle silk gland of normal silkworm and pCkG-CkR, E: fluorescence result of middle silk gland and cocoon shell of transgenic silkworm pG-R, pCkG-CkR, scale: 2 mm; F: detection of recombinant protein RFP and EGFP in cocoon of transgenic silkworm pG-R, pCkG-CkR, G: analysis of the content of recombinant protein RFP and EGFP in transgenic silkworm pCkG-CkR cocoon.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

The materials used in the practice of the invention are as follows:

cell line: spodoptera litura Sf9 ovarian cell line and silkworm embryo BmE cell. Both Sf9 cells and BmE cells were cultured using medium (Gibco) containing 10% fetal bovine serum (Gibco);

a target gene: the red fluorescent protein gene (DsRed), the green fluorescent protein gene (EGFP), the fusion genes DsRed-P2A-CkEGFP, CkEGFP-P2A-CkDsRed and EGFP-P2A-DsRed are synthesized by Kinry;

silkworm strain: non-diapauzing variety D9L.

Construction of transient expression vector and transgenic expression vector

Design rules according to Kozak sequence: base A, U, G in AUG is labeled as 1, 2, and 3, respectively, (1) the preferred base at position 4 is G; (2) the flanking sequence of the 5' end of AUG within about 15bp does not contain a base T; (3) at positions-3, -6 and-9, G is a biased base; (4) except for positions-3, -6 and-9, C is a favored base over the entire flanking sequence region. Thus, a classical Kozak sequence-accaccatgg- (Ck) (SEQ ID NO.39) was designed. Then, Ck was added to the N-terminus of the DsRed gene, and a Kozak sequence, designated-ACCACCATGG-, was constructed from the A4 promoter, Hr3 enhancer, and according to the classical Kozak sequence rule. Sk is a Kozak sequence at the N-terminal of the silkworm endogenous sericin 1 gene, and the sequence is-accgccaacatgg- (SEQ ID NO. 40). P2A has been optimized via a GSG linker (SEQ ID NO. 38).

Then, fusion genes DsRed-BmCPV2A (30) -EGFP, DsRed-BmCPV2A (30) -CkEGFP, DsRed-BmCPV2A (30) -SkEGFP, DsRed-P2A-CkEGFP, EGFP-P2A-DsRed and CkEGFP-P2A-CkDsRed were assembled by an overlap PCR method using the primers shown in Table 1. Wherein the fusion gene DsRed-BmCPV2A (30) -EGFP is assembled by SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.7 and SEQ ID NO. 8; the fusion gene DsRed-BmCPV2A (30) -CkEGFP, SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.11 and SEQ ID NO.12 is assembled by SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.9 and SEQ ID NO.10 to form the fusion gene DsRed-BmCPV2A (30) -SkEGFP; the fusion gene EGFP-P2A-DsRed is assembled by SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.13 and SEQ ID NO. 14; the fusion gene DsRed-P2A-CkEGFP is assembled by SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.15 and SEQ ID NO. 16; the fusion gene CkEGFP-P2A-CkDsRed is assembled by SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.17 and SEQ ID NO. 18.

And (2) carrying out double enzyme digestion by BamHI and NotI, and then recovering a target fragment, wherein the 1180PSL transient expression vector obtained after subcloning comprises 1180PSL (Hr 3Ser1DsRed Ser1 PA) or 1180PSL (Hr 3Ser1EGFP Ser1 PA) before subcloning: 1180PSL [ Hr3Ser1DsRed Ser1PA ], 1180PSL [ Hr3Ser1EGFP Ser1PA ], 1180PSL [ Hr3Ser1DsRed-BmCPV2A (30) -EGFP Ser1PA ], 1180PSL [ Hr3Ser1DsRed-BmCPV2A (30) -CkEGFP Ser1PA ], 1180PSL [ Hr3Ser1DsRed-BmCPV2A (30) -SkEGFP Ser1PA ], 1180PSL [ Hr3Ser1DsRed-P2A-EGFP Ser1PA ], 1180PSL [ Hr3Ser1EGFP 1-P2A-DsRed Ser1PA ] and 1180PSL [ Hr3Ser1DsRed-P2A-CkEGFP 1PA ]. BmCPV2A (30) is a 2A sequence derived from Bombyx mori cytoplasmic polyhedrosis virus and contains 30 amino acids (SEQ ID NO. 37).

The fusion genes DsRed-P2A-CkEGFP, CkEGFP-P2A-CkDsRed and EGFP-P2A-DsRed are subcloned for two times, and finally, the obtained transgenic expression vector comprises: piggyBac [3XP3DsRedSV 30; hr3Ser1DsRed-P2A-CkEGFP Ser1PA ], piggyBac [3XP3DsRed SV 30; hr3Ser1EGFP-P2A-DsRed Ser1PA ] and piggyBac [3XP3DsRed SV 30; hr3Ser1CkEGFP-P2A-CkDsRed Ser1PA ].

TABLE 1 primers used in vector construction

Second, cell transfection

And (3) after the constructed plasmid is converted again, selecting a single bacterial plaque and shaking the bacteria for 12h, taking 10mL of bacterial liquid, extracting the plasmid by using a Qiagen ultrapure kit, and measuring the purity of the plasmid by using a DNA concentration meter, wherein the 260/280 ratio is 1.8-1.9 for later use. SF9 and BmE cells were plated evenly on 24-well plates using Grace medium or Tc medium containing 10% fetal bovine serum, at a cell density of about 80%, and cultured overnight. Cells were washed 3 times with non-antibody, serum-free Grace medium or Tc medium, and 200. mu.l of non-antibody, serum-free Grace medium or Tc medium was added for future use. Preparation of plasmid transfection solution: after 1.5. mu.g of transfection plasmid is fully and uniformly mixed with 900. mu.l of non-antibody and serum-free Grace culture medium or Tc culture medium, 2.5. mu.l of transfection reagent X-Tree is added, uniformly mixed and shaken, and after standing for 30min, 300. mu.l of transfection reagent X-Tree is added into the cell wells for standby, and the total volume is 500. mu.l. After culturing for 6-8 hours, washing the cells for 3 times by using a non-antibody and serum-free culture medium or a Tc culture medium, then adding 500 mul Grace complete culture medium or Tc complete culture medium, and continuously culturing for 3 days.

Cultivation of transgenic silkworms

Feeding a polytropic silkworm variety D9L through high-quality mulberry leaves until the silkworm variety is cocooned, mating the silkworm variety D9L for 6-8 hours in a selfing mode after pupal period moth transformation, and placing a female moth at 4 ℃ for later use; preparation of injection plasmid: preparing a mixed solution of the plasmid to be injected and the Help plasmid containing Piggybab transposase according to the mass ratio of 1:1 for later use; the mother moths were removed and the plasmid mixture was injected into the eggs within 2h of birth under an SZX16 microscope (Olympus) using a TransferMan NK2 micromanipulator and a Femto Jet 5247 microsyringe (Eppendorf). Placing the injected silkworm eggs at the temperature of 25 ℃ and the relative humidity of 90% for incubation for 11 days to obtain G0 generation ant silkworms, carefully feeding fresh mulberry leaves to the cocooning stage, and carrying out selfing seed production after moth formation to obtain G1 generation ant silkworms. After the G1 generation silkworm embryo grows for 6 days, screening the eyes and nerve tissues of the silkworm embryo by using an SZX12 fluorescence stereomicroscope (Olympus), and obtaining the positive transgenic silkworm strain when the green fluorescence is presented.

Detection of target proteins

Extraction of cell protein sample: the medium was discarded, and 1mL of PBS (135mM NaC, 2.7mM KCl, 1.5mM KH) was used₂PO₄And 8mM K₂HPO₄pH7.4), adding 150 μ l of newly-prepared RIPA lysate containing protease inhibitor PSPF, placing on ice for 30min, collecting cells, centrifuging at 13400rpm for 5min, and taking supernatant as cell protein sample;

extracting a middle silk gland protein sample of the silkworm: dissecting middle silk gland of Bombyx mori, cutting, soaking in PBS, and extracting at 4 deg.C overnight. Centrifuging the extract at 13400rpm for 5min, and taking the supernatant as a middle silk gland protein sample of the silkworm;

extracting a cocoon shell protein sample: and (4) crushing the cocoon shells by using a crusher. 20mg of cocoon powder was extracted by shaking with 1mL of protein extract (8M urea, 25mM Tris-HCl, pH7.0) at 80 ℃ for 30 min. Centrifuging the extracting solution at 13400rpm for 5min, and taking the supernatant as a cocoon shell protein sample;

SDS-PAGE and Western Blotting: after the cell protein sample, the silkworm middle silk gland protein sample and the cocoon shell protein sample are subjected to protein concentration (Beyotime) determination by using an enhanced BCA protein determination kit, protein samples with equal mass are subjected to SDS-PAGE electrophoresis, and target protein is detected by adopting Coomassie brilliant blue staining and Western blotting. After the electrophoresis was completed, the protein sample was transferred to a PVDF membrane by a membrane transfer apparatus, and after blocking with 5% skim milk powder, applying primary antibodies (anti-RFP antibody, anti-EGFP antibody, anti-HSA antibody, anti-FGF 1 antibody, anti-VgR antibody, or anti-Tubulin antibody), 5 times of PBST washing, applying secondary antibodies (anti-mouse or rabbit IgG antibody), and 5 times of PBST washing, bands on the membrane were visualized using an ECL Western Blotting detection reagent (Amersham Biosciences), and the exposure mode was automatic exposure. And analyzing the intensity of the Western blotting band by using ImageJ software, and converting the intensity into a numerical value.

Fifth, observation of fluorescence signal

Red fluorescence signals and green fluorescence signals of SF9, BmE and bnn cells in a 24-well plate were directly observed and photographed using Olympus SZX12 fluorescence stereomicroscope (Olympus). The exposure time of the red fluorescence signal is 15ms, and the exposure time of the green fluorescence signal is 100 ms; directly observing and photographing red fluorescent signals, green red fluorescent signals and green fluorescent signals in the middle silk gland and cocoon shell of the silkworm on the seventh day by using an Olympus SZX12 fluorescent stereomicroscope. The exposure time for the red fluorescence signal was 30ms and the exposure time for the green fluorescence signal was 80 ms.

Sixth, quantitative PCR

Cell sample RT-PCR method: cells were harvested 3 days after transfection of the transient expression vector, and total RNA was extracted from the cells using a total RNA extraction kit (Omega). A quantity of RNA was reverse transcribed into cDNA template using a reverse transcription kit (Omega). Expression levels of DsRed gene and EGFP gene in each sample were measured by SYBR Premix extAQ kit (Takara) in ABI Fast 7000 quantitative PCR instrument (Applied Biosystems). Sw22934 was used as an internal reference gene.

The RT-PCR method of the middle silk gland of the transgenic silkworm comprises the following steps: dissecting middle silk gland of five-instar silkworm, and extracting total RNA by using the total RNA extraction kit. Reverse transcription of 2ug of RNA into cDNA was performed using a reverse transcription kit. mRNA of endogenous sericin 1 gene (Ser1), DsRed and EGFP gene in the sample cDNA was detected by SYBR Premix extAQ kit (Takara) in ABI Fast 7000 quantitative PCR instrument (Applied Biosystems) (Table 2).

TABLE 2 primers used in RT-PCR experiments

The above-mentioned primer^1-7The quantitative PCR primers are respectively used for detecting genes DsRed, EGFP, DsRed-2A-CkEGFP, EGFP-2A-DsRed, CkEGFP-2A-CkDsRed, Ser1 and SW 22934.

Seventh, experimental results

Ck enhancement of expression efficiency of downstream genes by increasing their translation efficiency

By adding Ck to the N-terminal of the DsRed gene, a transient expression vector Ck-DsRed (SEQ ID NO.32) regulated by the A4 promoter, Hr3 enhancer and Ser1PA terminator sequence and a control expression vector DsRed (FIG. 1, A) for normally expressing the DsRed gene were constructed. After transfection of Sf9 cells, it was found that the red fluorescence signal of Sf9 cells was significantly increased in the Ck-DsRed group to which Ck was added to the N-terminus of the DsRed gene, as compared to Sf9 cells of the DsRed group (FIG. 1, B). The quantitative PCR result shows that the expression of DsRed gene in the cells of DsRed and Ck-DsRed group has no obvious difference on mRNA level (figure 1, C); western blotting results show that the content of RFP protein in Sf9 cells of the Ck-DsRed group is obviously higher than that in Sf9 cells of the DsRed group, the content of RFP is improved by 1.8 times (figure 1, D-E), and the Ck can enhance the expression level of genes by improving the translation efficiency of downstream genes.

Ck enhancement of expression level of BmCPV2A (30) downstream EGFP Gene

To verify whether Ck could effectively improve the expression efficiency of 2A downstream genes, BmCPV2A (30) was selected as the target 2A sequence, since previous studies have demonstrated that BmCPV2A (30) could not effectively express its downstream EGFP gene. In order to compare the efficiency of Ck and Sk in enhancing the expression of EGFP gene downstream of BmCPV2A (30), Ck and Sk were fused to the fusion genes DsRed-CkEGFP and DsRed-SkEGFP which are connected to BmCPV2A (30), respectively, to construct corresponding transient expression vectors BmCPV2A (30) + Ck (SEQ ID NO.34) and BmCPV2A (30) + Sk (SEQ ID NO.35) (FIG. 2, A), and BmE cells were transfected. RT-PCR results showed no significant difference in the transcript levels of 3 DsRed-2A-EGFP fusion genes after transfection of transient expression vectors BmCPV2A (30) (SEQ ID NO.33), BmCPV2A (30) + Ck and BmCPV2A (30) + Sk (FIG. 2, B). Fluorescence results of Sf9 cells after transfection showed no difference in red fluorescence signals of Sf9 cells in BmCPV2A (30), BmCPV2A (30) + Ck and BmCPV2A (30) + Sk groups, but green fluorescence signals of bf 9 cells in BmCPV2A (30) + Ck and BmCPV2A (30) + Sk groups were significantly stronger than those of BmCPV2A (30) groups, and green fluorescence signals of Sf9 cells in BmCPV2A (30) + Ck groups were strongest (fig. 2, C). Western blotting results also showed that there was no significant difference in the expression levels of RFP in BmCPV2A (30), BmCPV2A (30) + Ck and BmCPV2A (30) + Sk group Sf9 cells, and that the expression levels of EGFP in BmCPV2A (30) + Ck and BmCPV2A (30) + Sk group Sf9 cells were significantly higher than that in BmCPV2A (30) group (FIG. 2, D-F). The above results indicate that, at the Sf9 cell level, the kozak sequence can be optimized to improve the expression level of BmCPV2A (30) downstream EGFP gene by enhancing the translation efficiency, and Ck is more efficient than Sk.

Equivalently recombining and expressing DsRed gene and EGFP gene in middle silk gland of silkworm by Ck-optimized silkworm silk gland 2A polygene expression system

In order to verify that Ck can enhance the expression efficiency of 2A downstream genes at the silkworm individual level, a fusion gene DsRed-P2A-CkEGFP (R-CkG) is assembled, a silkworm transgenic expression vector (SEQ ID NO.36) is constructed, and a transgenic silkworm strain (FIG. 3, A) is obtained. Generally, 35G 0 generation silkworm silkworms were obtained from 400 silkworm eggs injected, and 5G 1 generation moth rings, of which 2 positive moth rings were obtained after careful rearing and selfing seed production (Table 3). The transgenic line with the highest expression level of foreign proteins RFP and EGFP in the transgenic cocoon shell is named as pR-CkG. RT-PCR results show that the transcription of the fusion gene R-CkG is successfully detected in the middle silk gland of the transgenic silkworm pR-CkG, and the transcription level is 72.7 percent relative to the silkworm endogenous Ser1 gene (figure 3, C-D); the results of the fluorescence signals showed that the middle silk gland of transgenic silkworm pR-CkG exhibited strong and specific red and green fluorescence signals relative to normal silkworms (fig. 3, B); SDS-PAGE showed that recombinant expression of the disrupted RFP and EGFP proteins was detected in the middle silk gland of transgenic silkworm pR-CkG (FIG. 3, E). The result shows that P2A effectively mediates the recombinant expression and breakage of DsRed gene and EGFP gene in the fusion gene R-CkG. After analyzing the content of RFP and EGFP in the middle silk gland of transgenic silkworm pR-CkG, the content of RFP and EGFP is 0.18 +/-0.03% and 0.23 +/-0.04% respectively, and no significant difference is found (figure 3, F), compared with the content of RFP in transgenic silkworm pR-G which is far higher than the content of EGFP, the Ck can also effectively enhance the expression efficiency of the downstream EGFP gene of P2A in the level of silkworm individuals.

TABLE 3 transgenic silkworm pR-CkG, pG-R and pCkG-CkR transgenic injection statistics

Ck optimization for improving efficiency of recombinant expression of exogenous target gene of bombyx mori silk gland 2A polygene expression system in middle silkgland and silk of bombyx mori

In order to verify whether the Ck is added into a silkworm silk gland 2A polygene expression system to improve the expression level of the gene, transgenic silkworm strains of transgenic fusion genes EGFP-2A-DsRed (G-R) and CkEGFP-P2A-CkDsRed (CkG-CkR) are respectively constructed (figure 4, A-B and table 3). Through preliminary screening, the gene strain with the highest expression quantity of recombinant proteins RFP and EGFP in the transgenic cocoon shells is named as pG-R, pCkG-CkR.

RT-PCR results show that the expression of the fusion gene CkG-CkR is successfully detected in the middle silk gland of the transgenic silkworm pCkG-CkR, and the expression level is about 55.3 percent of the expression level of the endogenous Ser1 gene of the silkworm (figure 4, C-D). Fluorescence results show that strong and specific red and green fluorescence signals appear in the middle silk gland and cocoon shell of the transgenic silkworm pG-R, pCkG-CkR (figure 4, E); SDS-PAGE results showed that disrupted RFP and EGFP proteins were successfully detected in transgenic silkworm pG-R, pCkG-CkR cocoon shells (FIG. 4, F). The DsRed gene and the EGFP gene in the fusion gene G-R, CkG-CkR are successfully expressed and secreted into cocoon shells. In addition, compared with the fluorescence signals in the middle silk gland and the cocoon shell of the transgenic silkworm pG-R, the middle silk gland and the cocoon shell of the transgenic silkworm pCkG-CkR present stronger red fluorescence signals and green fluorescence signals (figure 4, E), the content of RFP and EGFP proteins in the cocoon shell of the transgenic silkworm pCkG-CkR is also obviously higher than that of the cocoon shell of the transgenic silkworm pG-R, and is improved by about 50 percent (figure 4, F), and the expression efficiency of genes can be obviously improved by introducing Ck at the level of the transgenic silkworm individual. More importantly, the contents of RFP and EGFP proteins in the cocoon shell of the transgenic silkworm pCkG-CkR are respectively 0.36 +/-0.03 percent and 0.28 +/-0.07 percent, and no obvious difference exists (figure 4, G).

Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.

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<210> 13

<211> 52

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

atccgggccc catgcgcttc gtattatgct gtaccctgat tgctttggct gc 52

<210> 14

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

caccatggtg gtggggcccg gattctcttc gacgtctcca gcct 44

<210> 15

<211> 61

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

atccgggccc caccaccatg gtgcgcttcg tattatgctg taccctgatt gctttggctg 60

c 61

<210> 16

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

caccatggtg gtggggcccg gattctcttc gacgtctcca gcct 44

<210> 17

<211> 61

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

atccgggccc caccaccatg gtgcgcttcg tattatgctg taccctgatt gctttggctg 60

c 61

<210> 18

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

cctacgaagg tcacaacaca gtc 23

<210> 19

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

ttttgtaatc aggtatgtca gcg 23

<210> 20

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

cattatcaac agaacacccc cat 23

<210> 21

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

tcctgctgct gtaacgaact ctaa 24

<210> 22

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

cgccaccatt tgttcctc 18

<210> 23

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

tgagggcagc caaagcaa 18

<210> 24

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

cagcaggaat caccttagga atg 23

<210> 25

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

tgagggcagc caaagcaa 18

<210> 26

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

cagcaggaat caccttagga atg 23

<210> 27

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

tgagggcagc caaagcaa 18

<210> 28

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

atctgaagac ggtttctggt ggt 23

<210> 29

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

aactgcctga agtggttgtg c 21

<210> 30

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

ttcgtactgg ctcttctcgt 20

<210> 31

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

caaagttgat agcaattccc t 21

<210> 32

<211> 2711

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

cagcgtcgtg aaaagaggca atgacaaata caaaacgacg tatgagcaga cccgtcgcca 60

agacgggtct acctctaaga tgatgtcatt tgttttttaa aactaactcg ctttacgagt 120

agaattctac gtgtaaaaca taatcaagag atgatgtcat ttgtttttca aaaccaaact 180

cgctttacga gtagaattct acgtgtaaaa cacaatcaaa agatgatgtc attcgttttt 240

caaaaccgaa tttaagaaat gatgtcattt gtttttcaaa accaaactcg ctttacgagc 300

agaattctac gtgtaaaaca caatcaagag atgatgtcat ttgtttttca aaactgaatg 360

atgtcatttg tttttcaaaa ctaaacttgc tttgcgagta gaattctacg tgtaaaacac 420

agtcaagaga tgatgtcatt tgtttttcaa aactgaaccg gctttacgag tagaattcta 480

cttgtaaaac ataatcaaga gatgatgtca tttgtttttc aaaactgaac tggctttacg 540

agtagaattc tacgtgtaaa acataatcaa gagatgatgt catcattaaa ctgatgtcat 600

tttatacacg attgttaaca tgtttaataa tgactaattt gtttttccaa attaaactcg 660

ctttacgagt agaattctac ttgtaacgca cgattaagta tgaatcataa gctgatgtca 720

tttgttttcg acataaaatg tttatacaat ggaatcttct tgtaaattat ccaaataata 780

taatttatcc gattctacgt tacatttaaa ttcgttgtta tcgtacaatt cttcaggaca 840

cgccatgtat tggtcatttt tagcgtgcaa ccaacgattg tatttgacgc cgtcgttgga 900

ttgcgtgttc aggttggcgt acacgtgact gggcacggct tctttttcca tgggacgtcg 960

acgaaaacag cacacacact acataccatg tatttgacgc acacacgcat gtatactatt 1020

tattgtcaaa cttttgttct tgacgtctgt gttcaaactg agaatagatt aaatattgtt 1080

tgtctttatt aatatttttt aatagtgtag tcttggcgaa atttgtgatt ataaaagtat 1140

aaaatacaat cataatagtg tacgaactta caattccaat taattatagt cgaatttcga 1200

ctactgcggg acctctagta ttaataattc tctttaaaaa aaaacagagc atcaaatact 1260

gcacaaatgt caagcgggtc tcaacgagcc atgaataaat tagaaatcaa ttaataacat 1320

aaaataggca aacaaaataa aaccatttac atagagaacg tttgttgaac aaaaacaata 1380

acttgtatac attgtttgca caaatgtttg aagcgaaaat ttattactct ctacgtaagc 1440

ttgatcaaac ttcgttttcg tataaaacgc gttggcccaa ccactttggc atagtcgtct 1500

tatcatcggg tctctaagga tcaagcgatc caaagaccgc caacatgcgt ttcgttctgt 1560

gctgcacttt gattgcgttg gctgcgctca gcgtaaaagc cttcggtcac caccccggca 1620

atcgagatac aggatccacc accatggtta gaagcagcaa aaatgttatc aaagagttta 1680

tgcgtttcaa agtgagaatg gaaggcacag ttaatggaca cgagttcgag atagaaggag 1740

agggagaagg ccgcccctac gaaggtcaca acacagtcaa attgaaagta actaagggtg 1800

gacctttacc attcgcgtgg gacattctta gtccacaatt tcagtatggc tcaaaagtgt 1860

acgttaagca tcccgctgac atacctgatt acaaaaagct ttccttcccg gaaggtttta 1920

aatgggagcg tgtgatgaac ttcgaggacg gcggtgtggt tacagttact caagactcat 1980

ctctccagga tggatgcttc atctacaagg tgaagttcat cggcgttaat tttccatccg 2040

atggtcctgt gatgcaaaag aaaacaatgg gctgggaagc ttcgactgag cgtctgtacc 2100

ctagggacgg agtcttgaaa ggcgaaatcc acaaggccct taaactgaag gatggaggcc 2160

attatctggt agagttcaag agcatttaca tggcaaaaaa gcccgtccaa ctccctggtt 2220

actactacgt agactctaag ctcgatataa ccagccacaa tgaagactat acgatcgttg 2280

agcagtacga aagaacagag ggacgccacc atttgttcct ctaagcggcc gctacaacta 2340

aacacgactt ggagtattcc ttgtagtgtt taagatttta aatcttactt aatgacttcg 2400

aacgatttta acgataactt tctctttgtt taactttaat cagcatacat aaaaagcccc 2460

ggttttgtat cgggaagaaa aaaaatgtaa ttgtgttgcc tagataataa acgtattatc 2520

aaagtgtgtg gttttccttt accaaagacc cctttaagat gggcctaatg ggcttaagtc 2580

gagtcctttc cgatgtgtta aatacacatt tattacactg atgcgtcgaa tgtacacttt 2640

taataggata gctccactaa aaattatttt atttatttaa tttgttgcac caaaactgat 2700

acattgacga a 2711

<210> 33

<211> 3602

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

cagcgtcgtg aaaagaggca atgacaaata caaaacgacg tatgagcaga cccgtcgcca 60

agacgggtct acctctaaga tgatgtcatt tgttttttaa aactaactcg ctttacgagt 120

agaattctac gtgtaaaaca taatcaagag atgatgtcat ttgtttttca aaaccaaact 180

cgctttacga gtagaattct acgtgtaaaa cacaatcaaa agatgatgtc attcgttttt 240

caaaaccgaa tttaagaaat gatgtcattt gtttttcaaa accaaactcg ctttacgagc 300

agaattctac gtgtaaaaca caatcaagag atgatgtcat ttgtttttca aaactgaatg 360

atgtcatttg tttttcaaaa ctaaacttgc tttgcgagta gaattctacg tgtaaaacac 420

agtcaagaga tgatgtcatt tgtttttcaa aactgaaccg gctttacgag tagaattcta 480

cttgtaaaac ataatcaaga gatgatgtca tttgtttttc aaaactgaac tggctttacg 540

agtagaattc tacgtgtaaa acataatcaa gagatgatgt catcattaaa ctgatgtcat 600

tttatacacg attgttaaca tgtttaataa tgactaattt gtttttccaa attaaactcg 660

ctttacgagt agaattctac ttgtaacgca cgattaagta tgaatcataa gctgatgtca 720

tttgttttcg acataaaatg tttatacaat ggaatcttct tgtaaattat ccaaataata 780

taatttatcc gattctacgt tacatttaaa ttcgttgtta tcgtacaatt cttcaggaca 840

cgccatgtat tggtcatttt tagcgtgcaa ccaacgattg tatttgacgc cgtcgttgga 900

ttgcgtgttc aggttggcgt acacgtgact gggcacggct tctttttcca tgggacgtcg 960

acgaaaacag cacacacact acataccatg tatttgacgc acacacgcat gtatactatt 1020

tattgtcaaa cttttgttct tgacgtctgt gttcaaactg agaatagatt aaatattgtt 1080

tgtctttatt aatatttttt aatagtgtag tcttggcgaa atttgtgatt ataaaagtat 1140

aaaatacaat cataatagtg tacgaactta caattccaat taattatagt cgaatttcga 1200

ctactgcggg acctctagta ttaataattc tctttaaaaa aaaacagagc atcaaatact 1260

gcacaaatgt caagcgggtc tcaacgagcc atgaataaat tagaaatcaa ttaataacat 1320

aaaataggca aacaaaataa aaccatttac atagagaacg tttgttgaac aaaaacaata 1380

acttgtatac attgtttgca caaatgtttg aagcgaaaat ttattactct ctacgtaagc 1440

ttgatcaaac ttcgttttcg tataaaacgc gttggcccaa ccactttggc atagtcgtct 1500

tatcatcggg tctctaagga tcaagcgatc caaagaccgc caacatgcgt ttcgttctgt 1560

gctgcacttt gattgcgttg gctgcgctca gcgtaaaagc cttcggtcac caccccggca 1620

atcgagatac aggatccatg gttagaagca gcaaaaatgt tatcaaagag tttatgcgtt 1680

tcaaagtgag aatggaaggc acagttaatg gacacgagtt cgagatagaa ggagagggag 1740

aaggccgccc ctacgaaggt cacaacacag tcaaattgaa agtaactaag ggtggacctt 1800

taccattcgc gtgggacatt cttagtccac aatttcagta tggctcaaaa gtgtacgtta 1860

agcatcccgc tgacatacct gattacaaaa agctttcctt cccggaaggt tttaaatggg 1920

agcgtgtgat gaacttcgag gacggcggtg tggttacagt tactcaagac tcatctctcc 1980

aggatggatg cttcatctac aaggtgaagt tcatcggcgt taattttcca tccgatggtc 2040

ctgtgatgca aaagaaaaca atgggctggg aagcttcgac tgagcgtctg taccctaggg 2100

acggagtctt gaaaggcgaa atccacaagg cccttaaact gaaggatgga ggccattatc 2160

tggtagagtt caagagcatt tacatggcaa aaaagcccgt ccaactccct ggttactact 2220

acgtagactc taagctcgat ataaccagcc acaatgaaga ctatacgatc gttgagcagt 2280

acgaaagaac agagggacgc caccatttgt tcctcagaac agcgttcgat ttccagcagg 2340

acgtttttcg ctctaattat gacctactaa agttgtgcgg tgatatcgag tctaatcctg 2400

gacctatggt gcgcttcgta ttatgctgta ccctgattgc tttggctgcc ctcagtgtca 2460

aggcctttgg tcaccatcca ggaaaccgtg atacaatggt atcaaaaggt gaagagctct 2520

tcactggagt cgtaccgatt ttagtcgaac ttgacggcga tgtaaatggt cacaagttta 2580

gcgtgtccgg cgagggtgaa ggagatgcta cctatggtaa attaacgctt aagttcatat 2640

gcacaactgg aaaattgcct gtgccatggc cgaccctggt taccacgttg acgtggggag 2700

ttcaatgttt ctctagatac cccgaccaca tgaaacagca tgatttcttt aagagcgcca 2760

tgcctgaagg atatgtccaa gagcgtacaa tcttctttaa agacgatggc aactacaaga 2820

ccagggcaga agtgaaattc gagggcgaca cgcttgttaa cagaatagaa ctgaaaggta 2880

tcgactttaa agaagacgga aacatcctcg gccacaagct cgaatacaac tacatctctc 2940

ataacgtgta cataactgca gataagcaaa agaacggtat caaagcgaat ttcaagatca 3000

ggcacaacat tgaggacgga agcgtgcagc tggctgatca ttatcaacag aacaccccca 3060

ttggcgacgg tccagttctc ttaccggata atcactactt gtcgacgcag agtgccctct 3120

caaaagaccc taatgaaaag agagatcata tggttctttt agagttcgtt acagcagcag 3180

gaatcacctt aggaatggat gagttataca aataagcggc cgctacaact aaacacgact 3240

tggagtattc cttgtagtgt ttaagatttt aaatcttact taatgacttc gaacgatttt 3300

aacgataact ttctctttgt ttaactttaa tcagcataca taaaaagccc cggttttgta 3360

tcgggaagaa aaaaaatgta attgtgttgc ctagataata aacgtattat caaagtgtgt 3420

ggttttcctt taccaaagac ccctttaaga tgggcctaat gggcttaagt cgagtccttt 3480

ccgatgtgtt aaatacacat ttattacact gatgcgtcga atgtacactt ttaataggat 3540

agctccacta aaaattattt tatttattta atttgttgca ccaaaactga tacattgacg 3600

aa 3602

<210> 34

<211> 3608

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

cagcgtcgtg aaaagaggca atgacaaata caaaacgacg tatgagcaga cccgtcgcca 60

agacgggtct acctctaaga tgatgtcatt tgttttttaa aactaactcg ctttacgagt 120

agaattctac gtgtaaaaca taatcaagag atgatgtcat ttgtttttca aaaccaaact 180

cgctttacga gtagaattct acgtgtaaaa cacaatcaaa agatgatgtc attcgttttt 240

caaaaccgaa tttaagaaat gatgtcattt gtttttcaaa accaaactcg ctttacgagc 300

agaattctac gtgtaaaaca caatcaagag atgatgtcat ttgtttttca aaactgaatg 360

atgtcatttg tttttcaaaa ctaaacttgc tttgcgagta gaattctacg tgtaaaacac 420

agtcaagaga tgatgtcatt tgtttttcaa aactgaaccg gctttacgag tagaattcta 480

cttgtaaaac ataatcaaga gatgatgtca tttgtttttc aaaactgaac tggctttacg 540

agtagaattc tacgtgtaaa acataatcaa gagatgatgt catcattaaa ctgatgtcat 600

tttatacacg attgttaaca tgtttaataa tgactaattt gtttttccaa attaaactcg 660

ctttacgagt agaattctac ttgtaacgca cgattaagta tgaatcataa gctgatgtca 720

tttgttttcg acataaaatg tttatacaat ggaatcttct tgtaaattat ccaaataata 780

taatttatcc gattctacgt tacatttaaa ttcgttgtta tcgtacaatt cttcaggaca 840

cgccatgtat tggtcatttt tagcgtgcaa ccaacgattg tatttgacgc cgtcgttgga 900

ttgcgtgttc aggttggcgt acacgtgact gggcacggct tctttttcca tgggacgtcg 960

acgaaaacag cacacacact acataccatg tatttgacgc acacacgcat gtatactatt 1020

tattgtcaaa cttttgttct tgacgtctgt gttcaaactg agaatagatt aaatattgtt 1080

tgtctttatt aatatttttt aatagtgtag tcttggcgaa atttgtgatt ataaaagtat 1140

aaaatacaat cataatagtg tacgaactta caattccaat taattatagt cgaatttcga 1200

ctactgcggg acctctagta ttaataattc tctttaaaaa aaaacagagc atcaaatact 1260

gcacaaatgt caagcgggtc tcaacgagcc atgaataaat tagaaatcaa ttaataacat 1320

aaaataggca aacaaaataa aaccatttac atagagaacg tttgttgaac aaaaacaata 1380

acttgtatac attgtttgca caaatgtttg aagcgaaaat ttattactct ctacgtaagc 1440

ttgatcaaac ttcgttttcg tataaaacgc gttggcccaa ccactttggc atagtcgtct 1500

tatcatcggg tctctaagga tcaagcgatc caaagaccgc caacatgcgt ttcgttctgt 1560

gctgcacttt gattgcgttg gctgcgctca gcgtaaaagc cttcggtcac caccccggca 1620

atcgagatac aggatccatg gttagaagca gcaaaaatgt tatcaaagag tttatgcgtt 1680

tcaaagtgag aatggaaggc acagttaatg gacacgagtt cgagatagaa ggagagggag 1740

aaggccgccc ctacgaaggt cacaacacag tcaaattgaa agtaactaag ggtggacctt 1800

taccattcgc gtgggacatt cttagtccac aatttcagta tggctcaaaa gtgtacgtta 1860

agcatcccgc tgacatacct gattacaaaa agctttcctt cccggaaggt tttaaatggg 1920

agcgtgtgat gaacttcgag gacggcggtg tggttacagt tactcaagac tcatctctcc 1980

aggatggatg cttcatctac aaggtgaagt tcatcggcgt taattttcca tccgatggtc 2040

ctgtgatgca aaagaaaaca atgggctggg aagcttcgac tgagcgtctg taccctaggg 2100

acggagtctt gaaaggcgaa atccacaagg cccttaaact gaaggatgga ggccattatc 2160

tggtagagtt caagagcatt tacatggcaa aaaagcccgt ccaactccct ggttactact 2220

acgtagactc taagctcgat ataaccagcc acaatgaaga ctatacgatc gttgagcagt 2280

acgaaagaac agagggacgc caccatttgt tcctcagaac agcgttcgat ttccagcagg 2340

acgtttttcg ctctaattat gacctactaa agttgtgcgg tgatatcgag tctaatcctg 2400

gacctaccac catggtgcgc ttcgtattat gctgtaccct gattgctttg gctgccctca 2460

gtgtcaaggc ctttggtcac catccaggaa accgtgatac aatggtatca aaaggtgaag 2520

agctcttcac tggagtcgta ccgattttag tcgaacttga cggcgatgta aatggtcaca 2580

agtttagcgt gtccggcgag ggtgaaggag atgctaccta tggtaaatta acgcttaagt 2640

tcatatgcac aactggaaaa ttgcctgtgc catggccgac cctggttacc acgttgacgt 2700

ggggagttca atgtttctct agataccccg accacatgaa acagcatgat ttctttaaga 2760

gcgccatgcc tgaaggatat gtccaagagc gtacaatctt ctttaaagac gatggcaact 2820

acaagaccag ggcagaagtg aaattcgagg gcgacacgct tgttaacaga atagaactga 2880

aaggtatcga ctttaaagaa gacggaaaca tcctcggcca caagctcgaa tacaactaca 2940

tctctcataa cgtgtacata actgcagata agcaaaagaa cggtatcaaa gcgaatttca 3000

agatcaggca caacattgag gacggaagcg tgcagctggc tgatcattat caacagaaca 3060

cccccattgg cgacggtcca gttctcttac cggataatca ctacttgtcg acgcagagtg 3120

ccctctcaaa agaccctaat gaaaagagag atcatatggt tcttttagag ttcgttacag 3180

cagcaggaat caccttagga atggatgagt tatacaaata agcggccgct acaactaaac 3240

acgacttgga gtattccttg tagtgtttaa gattttaaat cttacttaat gacttcgaac 3300

gattttaacg ataactttct ctttgtttaa ctttaatcag catacataaa aagccccggt 3360

tttgtatcgg gaagaaaaaa aatgtaattg tgttgcctag ataataaacg tattatcaaa 3420

gtgtgtggtt ttcctttacc aaagacccct ttaagatggg cctaatgggc ttaagtcgag 3480

tcctttccga tgtgttaaat acacatttat tacactgatg cgtcgaatgt acacttttaa 3540

taggatagct ccactaaaaa ttattttatt tatttaattt gttgcaccaa aactgataca 3600

ttgacgaa 3608

<210> 35

<211> 3611

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

cagcgtcgtg aaaagaggca atgacaaata caaaacgacg tatgagcaga cccgtcgcca 60

agacgggtct acctctaaga tgatgtcatt tgttttttaa aactaactcg ctttacgagt 120

agaattctac gtgtaaaaca taatcaagag atgatgtcat ttgtttttca aaaccaaact 180

cgctttacga gtagaattct acgtgtaaaa cacaatcaaa agatgatgtc attcgttttt 240

caaaaccgaa tttaagaaat gatgtcattt gtttttcaaa accaaactcg ctttacgagc 300

agaattctac gtgtaaaaca caatcaagag atgatgtcat ttgtttttca aaactgaatg 360

atgtcatttg tttttcaaaa ctaaacttgc tttgcgagta gaattctacg tgtaaaacac 420

agtcaagaga tgatgtcatt tgtttttcaa aactgaaccg gctttacgag tagaattcta 480

cttgtaaaac ataatcaaga gatgatgtca tttgtttttc aaaactgaac tggctttacg 540

agtagaattc tacgtgtaaa acataatcaa gagatgatgt catcattaaa ctgatgtcat 600

tttatacacg attgttaaca tgtttaataa tgactaattt gtttttccaa attaaactcg 660

ctttacgagt agaattctac ttgtaacgca cgattaagta tgaatcataa gctgatgtca 720

tttgttttcg acataaaatg tttatacaat ggaatcttct tgtaaattat ccaaataata 780

taatttatcc gattctacgt tacatttaaa ttcgttgtta tcgtacaatt cttcaggaca 840

cgccatgtat tggtcatttt tagcgtgcaa ccaacgattg tatttgacgc cgtcgttgga 900

ttgcgtgttc aggttggcgt acacgtgact gggcacggct tctttttcca tgggacgtcg 960

acgaaaacag cacacacact acataccatg tatttgacgc acacacgcat gtatactatt 1020

tattgtcaaa cttttgttct tgacgtctgt gttcaaactg agaatagatt aaatattgtt 1080

tgtctttatt aatatttttt aatagtgtag tcttggcgaa atttgtgatt ataaaagtat 1140

aaaatacaat cataatagtg tacgaactta caattccaat taattatagt cgaatttcga 1200

ctactgcggg acctctagta ttaataattc tctttaaaaa aaaacagagc atcaaatact 1260

gcacaaatgt caagcgggtc tcaacgagcc atgaataaat tagaaatcaa ttaataacat 1320

aaaataggca aacaaaataa aaccatttac atagagaacg tttgttgaac aaaaacaata 1380

acttgtatac attgtttgca caaatgtttg aagcgaaaat ttattactct ctacgtaagc 1440

ttgatcaaac ttcgttttcg tataaaacgc gttggcccaa ccactttggc atagtcgtct 1500

tatcatcggg tctctaagga tcaagcgatc caaagaccgc caacatgcgt ttcgttctgt 1560

gctgcacttt gattgcgttg gctgcgctca gcgtaaaagc cttcggtcac caccccggca 1620

atcgagatac aggatccatg gttagaagca gcaaaaatgt tatcaaagag tttatgcgtt 1680

tcaaagtgag aatggaaggc acagttaatg gacacgagtt cgagatagaa ggagagggag 1740

aaggccgccc ctacgaaggt cacaacacag tcaaattgaa agtaactaag ggtggacctt 1800

taccattcgc gtgggacatt cttagtccac aatttcagta tggctcaaaa gtgtacgtta 1860

agcatcccgc tgacatacct gattacaaaa agctttcctt cccggaaggt tttaaatggg 1920

agcgtgtgat gaacttcgag gacggcggtg tggttacagt tactcaagac tcatctctcc 1980

aggatggatg cttcatctac aaggtgaagt tcatcggcgt taattttcca tccgatggtc 2040

ctgtgatgca aaagaaaaca atgggctggg aagcttcgac tgagcgtctg taccctaggg 2100

acggagtctt gaaaggcgaa atccacaagg cccttaaact gaaggatgga ggccattatc 2160

tggtagagtt caagagcatt tacatggcaa aaaagcccgt ccaactccct ggttactact 2220

acgtagactc taagctcgat ataaccagcc acaatgaaga ctatacgatc gttgagcagt 2280

acgaaagaac agagggacgc caccatttgt tcctcagaac agcgttcgat ttccagcagg 2340

acgtttttcg ctctaattat gacctactaa agttgtgcgg tgatatcgag tctaatcctg 2400

gacctaccgc caacatggtg cgcttcgtat tatgctgtac cctgattgct ttggctgccc 2460

tcagtgtcaa ggcctttggt caccatccag gaaaccgtga tacaatggta tcaaaaggtg 2520

aagagctctt cactggagtc gtaccgattt tagtcgaact tgacggcgat gtaaatggtc 2580

acaagtttag cgtgtccggc gagggtgaag gagatgctac ctatggtaaa ttaacgctta 2640

agttcatatg cacaactgga aaattgcctg tgccatggcc gaccctggtt accacgttga 2700

cgtggggagt tcaatgtttc tctagatacc ccgaccacat gaaacagcat gatttcttta 2760

agagcgccat gcctgaagga tatgtccaag agcgtacaat cttctttaaa gacgatggca 2820

actacaagac cagggcagaa gtgaaattcg agggcgacac gcttgttaac agaatagaac 2880

tgaaaggtat cgactttaaa gaagacggaa acatcctcgg ccacaagctc gaatacaact 2940

acatctctca taacgtgtac ataactgcag ataagcaaaa gaacggtatc aaagcgaatt 3000

tcaagatcag gcacaacatt gaggacggaa gcgtgcagct ggctgatcat tatcaacaga 3060

acacccccat tggcgacggt ccagttctct taccggataa tcactacttg tcgacgcaga 3120

gtgccctctc aaaagaccct aatgaaaaga gagatcatat ggttctttta gagttcgtta 3180

cagcagcagg aatcacctta ggaatggatg agttatacaa ataagcggcc gctacaacta 3240

aacacgactt ggagtattcc ttgtagtgtt taagatttta aatcttactt aatgacttcg 3300

aacgatttta acgataactt tctctttgtt taactttaat cagcatacat aaaaagcccc 3360

ggttttgtat cgggaagaaa aaaaatgtaa ttgtgttgcc tagataataa acgtattatc 3420

aaagtgtgtg gttttccttt accaaagacc cctttaagat gggcctaatg ggcttaagtc 3480

gagtcctttc cgatgtgtta aatacacatt tattacactg atgcgtcgaa tgtacacttt 3540

taataggata gctccactaa aaattatttt atttatttaa tttgttgcac caaaactgat 3600

acattgacga a 3611

<210> 36

<211> 3596

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

cagcgtcgtg aaaagaggca atgacaaata caaaacgacg tatgagcaga cccgtcgcca 60

agacgggtct acctctaaga tgatgtcatt tgttttttaa aactaactcg ctttacgagt 120

agaattctac gtgtaaaaca taatcaagag atgatgtcat ttgtttttca aaaccaaact 180

cgctttacga gtagaattct acgtgtaaaa cacaatcaaa agatgatgtc attcgttttt 240

caaaaccgaa tttaagaaat gatgtcattt gtttttcaaa accaaactcg ctttacgagc 300

agaattctac gtgtaaaaca caatcaagag atgatgtcat ttgtttttca aaactgaatg 360

atgtcatttg tttttcaaaa ctaaacttgc tttgcgagta gaattctacg tgtaaaacac 420

agtcaagaga tgatgtcatt tgtttttcaa aactgaaccg gctttacgag tagaattcta 480

cttgtaaaac ataatcaaga gatgatgtca tttgtttttc aaaactgaac tggctttacg 540

agtagaattc tacgtgtaaa acataatcaa gagatgatgt catcattaaa ctgatgtcat 600

tttatacacg attgttaaca tgtttaataa tgactaattt gtttttccaa attaaactcg 660

ctttacgagt agaattctac ttgtaacgca cgattaagta tgaatcataa gctgatgtca 720

tttgttttcg acataaaatg tttatacaat ggaatcttct tgtaaattat ccaaataata 780

taatttatcc gattctacgt tacatttaaa ttcgttgtta tcgtacaatt cttcaggaca 840

cgccatgtat tggtcatttt tagcgtgcaa ccaacgattg tatttgacgc cgtcgttgga 900

ttgcgtgttc aggttggcgt acacgtgact gggcacggct tctttttcca tgggacgtcg 960

acgaaaacag cacacacact acataccatg tatttgacgc acacacgcat gtatactatt 1020

tattgtcaaa cttttgttct tgacgtctgt gttcaaactg agaatagatt aaatattgtt 1080

tgtctttatt aatatttttt aatagtgtag tcttggcgaa atttgtgatt ataaaagtat 1140

aaaatacaat cataatagtg tacgaactta caattccaat taattatagt cgaatttcga 1200

ctactgcggg acctctagta ttaataattc tctttaaaaa aaaacagagc atcaaatact 1260

gcacaaatgt caagcgggtc tcaacgagcc atgaataaat tagaaatcaa ttaataacat 1320

aaaataggca aacaaaataa aaccatttac atagagaacg tttgttgaac aaaaacaata 1380

acttgtatac attgtttgca caaatgtttg aagcgaaaat ttattactct ctacgtaagc 1440

ttgatcaaac ttcgttttcg tataaaacgc gttggcccaa ccactttggc atagtcgtct 1500

tatcatcggg tctctaagga tcaagcgatc caaagaccgc caacatgcgt ttcgttctgt 1560

gctgcacttt gattgcgttg gctgcgctca gcgtaaaagc cttcggtcac caccccggca 1620

atcgagatac aggatccatg gttagaagca gcaaaaatgt tatcaaagag tttatgcgtt 1680

tcaaagtgag aatggaaggc acagttaatg gacacgagtt cgagatagaa ggagagggag 1740

aaggccgccc ctacgaaggt cacaacacag tcaaattgaa agtaactaag ggtggacctt 1800

taccattcgc gtgggacatt cttagtccac aatttcagta tggctcaaaa gtgtacgtta 1860

agcatcccgc tgacatacct gattacaaaa agctttcctt cccggaaggt tttaaatggg 1920

agcgtgtgat gaacttcgag gacggcggtg tggttacagt tactcaagac tcatctctcc 1980

aggatggatg cttcatctac aaggtgaagt tcatcggcgt taattttcca tccgatggtc 2040

ctgtgatgca aaagaaaaca atgggctggg aagcttcgac tgagcgtctg taccctaggg 2100

acggagtctt gaaaggcgaa atccacaagg cccttaaact gaaggatgga ggccattatc 2160

tggtagagtt caagagcatt tacatggcaa aaaagcccgt ccaactccct ggttactact 2220

acgtagactc taagctcgat ataaccagcc acaatgaaga ctatacgatc gttgagcagt 2280

acgaaagaac agagggacgc caccatttgt tcctcagagc aaagcgcggt tccggagcga 2340

ctaacttttc gctgttgaaa caggctggag acgtcgaaga gaatccgggc cccaccacca 2400

tggtgcgctt cgtattatgc tgtaccctga ttgctttggc tgccctcagt gtcaaggcct 2460

ttggtcacca tccaggaaac cgtgatacaa tggtatcaaa aggtgaagag ctcttcactg 2520

gagtcgtacc gattttagtc gaacttgacg gcgatgtaaa tggtcacaag tttagcgtgt 2580

ccggcgaggg tgaaggagat gctacctatg gtaaattaac gcttaagttc atatgcacaa 2640

ctggaaaatt gcctgtgcca tggccgaccc tggttaccac gttgacgtgg ggagttcaat 2700

gtttctctag ataccccgac cacatgaaac agcatgattt ctttaagagc gccatgcctg 2760

aaggatatgt ccaagagcgt acaatcttct ttaaagacga tggcaactac aagaccaggg 2820

cagaagtgaa attcgagggc gacacgcttg ttaacagaat agaactgaaa ggtatcgact 2880

ttaaagaaga cggaaacatc ctcggccaca agctcgaata caactacatc tctcataacg 2940

tgtacataac tgcagataag caaaagaacg gtatcaaagc gaatttcaag atcaggcaca 3000

acattgagga cggaagcgtg cagctggctg atcattatca acagaacacc cccattggcg 3060

acggtccagt tctcttaccg gataatcact acttgtcgac gcagagtgcc ctctcaaaag 3120

accctaatga aaagagagat catatggttc ttttagagtt cgttacagca gcaggaatca 3180

ccttaggaat ggatgagtta tacaaataag cggccgctac aactaaacac gacttggagt 3240

attccttgta gtgtttaaga ttttaaatct tacttaatga cttcgaacga ttttaacgat 3300

aactttctct ttgtttaact ttaatcagca tacataaaaa gccccggttt tgtatcggga 3360

agaaaaaaaa tgtaattgtg ttgcctagat aataaacgta ttatcaaagt gtgtggtttt 3420

cctttaccaa agaccccttt aagatgggcc taatgggctt aagtcgagtc ctttccgatg 3480

tgttaaatac acatttatta cactgatgcg tcgaatgtac acttttaata ggatagctcc 3540

actaaaaatt attttattta tttaatttgt tgcaccaaaa ctgatacatt gacgaa 3596

<210> 37

<211> 90

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

agaacagcgt tcgatttcca gcaggacgtt tttcgctcta attatgacct actaaagttg 60

tgcggtgata tcgagtctaa tcctggacct 90

<210> 38

<211> 78

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

agagcaaagc gcggttccgg agcgactaac ttttcgctgt tgaaacaggc tggagacgtc 60

gaagagaatc cgggcccc 78

<210> 39

<211> 10

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

accaccatgg 10

<210> 40

<211> 13

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

accgccaaca tgg 13

Claims (1)

1. A method for realizing equivalent expression of two genes in silkworm silk glands by a multigene expression vector based on 2A lytic peptide is characterized in that: the polygene expression vector contains a fusion gene with a structure of Ck-target gene-2A-Ck-target gene, wherein 2A lytic peptide is used for connecting two target genes, and kozak sequences Ck are respectively added at the upstream of the two target genes; the 2A split peptide is pig enterovirus 2A split peptide optimized by a GSG joint, the sequence of the 2A split peptide is shown as SEQ ID NO.38, and the nucleotide sequence of Ck is shown as SEQ ID NO. 39; the expression of the fusion gene is regulated in the vector by the A4 promoter, Hr3 enhancer and Ser1PA terminator sequences.

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