CN101270366A - Method for Improving the Level of Exogenous Gene Expression in Transgenic Insect Cells - Google Patents

Abstract

The invention discloses a method for improving the expression of exogenous genes in insect transgenic cells. The neomycin resistance gene expression box, enhancer element and exogenous gene expression box controlled by an active promoter in insect cells are genetically manipulated. Cloned into the vector with the reporter gene based on the transposon piggyBAC factor, and then mixed with the helper plasmid expressing the transposase to transfect the insect cell line, and the transgenic insect cells were obtained through the segmental selection of G418. The transgenic cells are engineered cells with high-level expression of exogenous genes obtained through cell cloning technology combined with actual detection of exogenous gene expression levels. The method of the invention realizes high-level continuous expression of exogenous genes in transgenic insect cells, and the expression products are free from baculovirus pollution, have high biological safety, perfect post-processing and good naturalness.

Description

Method for Improving the Level of Exogenous Gene Expression in Transgenic Insect Cells

technical field

The invention relates to a transgene expression system, in particular to a method for increasing the expression level of exogenous genes in transgenic insect cells.

Background technique

There are two main insect baculovirus expression systems currently in use, one uses the alfalfa geometrid spodoptera nuclear polyhedrosis virus as a vector, and uses Fall Armyworm cells, cabbage caterpillar cells or insects as hosts; the other uses the silkworm nuclear polyhedrosis virus as a host. The body virus is used as the carrier, and the silkworm cell or silkworm worm and pupa are used as the expression host. Both expression systems use strong promoters such as polyhedrin gene or P10 gene or very early gene IE-1 of their respective viral vectors as regulatory elements for exogenous gene expression, by placing the exogenous target gene under the control of a strong promoter When the recombinant virus infects the host cell, a large amount of recombinant baculovirus replicates in the host and the cell. In the late stage of virus infection of the host, the transcripts of the foreign gene accumulate in large quantities and are highly expressed in a short period of time.

Compared with the above insect baculovirus expression vector system, the use of transformed cells to express foreign genes has its own advantages:

(1) Perfect post-translational processing, high and stable natural activity, sustainable passage, and high production efficiency;

(2) Expressed by transformed cells, baculovirus is not involved in the whole process, and the biological safety is relatively improved, and the exogenous gene is integrated into the cell genome, and there will be no phenomenon of affecting cell function due to virus infection. Therefore, the expression product It can be processed more efficiently, and if the serum-free insect cell culture technology and secretion expression technology are used, the expression product will be very easy to purify.

Usually, transformed cells stably expressing foreign genes can be obtained by antibiotic stress selection. There have been many reports on the use of G418 to screen mammalian transformed cells, but there are few reports on the expression of foreign genes through stably transformed insect cells. Jarvis et al. transfected sf-9 cells with a plasmid carrying the neo marker gene and the ie-1 (very early gene of the californica nuclear polyhedrosis virus) promoter to control the expression cassette of the β-galactosidase gene, and passed the G418 Pressure screening to obtain a pure line of transformed sf-9 cells, the cells after 50 passages (about 6 months) still retain the integrated plasmid sequence, and the cells after 100 passages can still express homogeneous β-galactosidase, but The expression level is low, about 1% of the baculovirus expression vector system (BEVS) (J Cell Biochem, 1990, 42: 181-191).

Recently, Invitrogen has developed a vector pIZT/V5-His that can stably express foreign genes in insect sf cells. The vector uses the ie-2 promoter of Orgyia pseudotsugata nuclear polyhedrosis virus to control the foreign Genes can be screened by Zeocin resistance markers to obtain cell lines stably expressing foreign genes.

In view of the superiority of using transgenic insect cells to express exogenous genes, it is of great significance to independently develop methods for improving the level of stably transformed insect cells expressing exogenous genes.

Contents of the invention

The purpose of the present invention is to provide a method for increasing the expression level of exogenous gene in transgenic insect cells.

In order to achieve the above object, the technical solution adopted in the present invention is: a method for improving the level of exogenous gene expression in transgenic insect cells, comprising the following specific steps:

(1) Constructing an active promoter X in the insect cell to control the vector of the exogenous gene;

(2) Construct the carrier pigA3-en with reporter gene based on piggyBAC transposon with enhancer element en;

(3) The vector obtained in the double enzyme digestion step (1), recovering the fragment of the exogenous gene controlled by the promoter X, and cloning it into the same double enzyme digested pigA3-en, to obtain a fragment with the enhancer en and the promoter X controlling the exogenous gene carrier;

(4) Constructing the recombinant vector of the active promoter Y controlling the neomycin resistance gene neo in insect cells;

(5) The vector obtained in step (4) of enzyme digestion, the fragment Y-Neo of the neomycin resistance gene controlled by the promoter Y is recovered, and cloned into the vector obtained in the step (3) of the same restriction enzyme digestion, to obtain the vector with the promoter X controlling the gene Source gene expression cassette, enhancer element, promoter Y control the recombinant transgenic vector of neomycin resistance gene and fluorescent protein reporter gene;

(6) Mix the recombinant transgenic vector obtained in step (5) with the helper plasmid expressing the transposase, transfect the insect cell line, and obtain the transgenic insect cell by segmental screening of G418, wherein the final concentration of G418 is 800-2000 μg/ml .

In the above-mentioned technical scheme, active promoters X and Y in insect cells are selected from the very early gene (ie-1) promoter of baculovirus, the silkworm actin (actin) A3 promoter, cytomegalovirus (Cytomegalovirus) One of the CMV promoters and insect heat shock protein (hsp) promoters, the promoter X and the promoter Y can be the same or different; the enhancer element en is selected from the first part of the silk fibroin light chain gene Containing subsequence—fiben, homologous region of baculovirus—one of hr3en; reporter gene can be a fluorescent protein gene; insect cells can be silkworm cells or cell lines established by other insect tissues.

The selection of the helper plasmid in the above technical solution is a conventional technique in this technical field, and those skilled in the art can select the corresponding helper plasmid according to the specific transgenic vector obtained at last.

In a preferred technical solution, the insect cells are silkworm cells, and the promoter is one of the very early gene (ie-1) promoter of baculovirus and the silkworm actin (actin) A3 promoter.

In the above technical scheme, the transposon piggyBAC factor, also known as IFP2, is a widely used insect transposon at present. It has been confirmed that it can play a transposition role in various insects, and can recombine and integrate foreign genes through transposition Into the insect genome, but according to research, piggyBac-mediated transgenic silkworm cell expression reporter gene level is low (Zhou Wenlin et al., Sericulture Science, 2007, 33(1): 30-35), and it is not useful based on The report of the transgenic carrier of piggyBAC transposon to improve the expression of exogenous gene in insect transgenic cells.

In the above technical solutions, an enhancer refers to a DNA sequence that increases the transcription frequency of genes linked to it. Enhancers increase transcription through a promoter. Effective enhancers can be located at the 5' end of the gene, or at the 3' end of the gene, and some can also be located in the intron of the gene. The effect of the enhancer is obvious, generally it can increase the gene transcription frequency by 10-200 times, and some can even be as high as thousands of times. The effect of the enhancer has nothing to do with the orientation of the enhancer, and even thousands of kb away from the target gene still has the enhanced effect.

The basic principle of the present invention is: after constructing the recombinant transgenic vector with promoter X to control exogenous gene expression cassette, enhancer element, promoter Y to control neomycin resistance gene and fluorescent protein reporter gene, and express transposase Mix the helper plasmids, transfect insect cell lines, and pass the segmental screening of G418. Since G418 has a killing effect on general cells, and the target plasmid for transfection has a NEO drug resistance gene, the integration of foreign genes can finally be obtained. At the same time, in order to increase the expression level of exogenous genes in transgenic insect cells, in the process of constructing the transgenic vector, the enhancer was cloned, and the piggyBAC transposon was used, and G418 can also be used for transfection Cellular stress makes it difficult for foreign genes to be lost during chromosome replication.

In order to further improve the expression level of exogenous genes in insect transgenic cells, further technical solutions can be adopted to obtain engineered cells with high levels of exogenous gene expression by combining the obtained transgenic cells with cell cloning technology and the actual detection of exogenous gene expression levels.

The invention can adopt secretion expression combined with insect cell serum-free fermentation and culture technology, and the preparation process of expression product is simple and low in cost.

Due to the use of the above-mentioned technical solutions, the present invention has the following advantages compared with the prior art:

1. Due to the use of transgenic insect cells to express foreign genes instead of insect baculovirus expression vector system, continuous expression of foreign genes can be achieved; the expression products are free of baculovirus contamination and have high biological safety; the expression products are post-processed Perfect and natural.

2. The present invention adds resistance genes that can be used for stress screening when transferring foreign genes, which not only improves the screening efficiency of transgenic cells, but also G418 can also put pressure on transfected cells, so that foreign genes are not easy to be in the process of chromosome replication lost.

3. The technical scheme and principle of the present invention are different from the technical principle of Jarvis (J Cell Biochem, 1990, 42: 181-191), and also different from a kind of exogenous gene vector pIZT developed by Invitrogen Company which can stably express foreign gene in insect sf cells The technical principle of /V5-His, and introduce the enhancer element into the vector, use the piggyBAC transposon, and through the cell cloning technology, combined with the actual detection of the expression level of the foreign gene, screen the engineered cells with a high level of expression of the foreign gene .

Detailed ways

The present invention will be further described below in conjunction with embodiment:

Embodiment one: a kind of method that improves transgenic silkworm cell to express human insulin-like growth factor-I (IGF-1 under the control of A3 promoter, the neo of the first intron fiben of silk protein light chain gene, IE-1 promoter control ), including the following steps:

1. Preparation of human insulin-like growth factor-I gene

According to the cDNA sequence (GenBank accession number M11568) of the published human insulin-like growth factor-I gene, the DNA sequence corresponding to the human insulin-like growth factor-I active peptide is artificially synthesized according to the routine:

TGGATATCACCATGggaccggagacgctctgcggggctgagctggtggatgctcttcagttcgtgtgtggagacagggggcttttatttcaacaagccccacagggtatggctccagcagtcggagggcgcctcagacaggcatcgtggatgagtgctgcttccggagctgtgatctaaggaggcaAGcaaggatgtattgcgcTCGAAT

In order to facilitate cloning and expression and increase the expression level, the initiation codon ATG and EcoRV restriction site were introduced at the 5' end, and the termination codon TAA and Xho I restriction site were introduced at the 3' end.

2. Construction of pBluescript II SK(+) recombinant plasmid with cDNA gene

The artificially synthesized DNA sequence was double-digested with EcoR V and Xho I, and the digested fragment was recovered by conventional methods, and ligated with the pBluescript II SK (+) plasmid (product of Stratagene) double-digested with EcoRV and Xho I. T ₄ DNA ligase is a product of GIBCO BRL Company, and the ligation condition is 16°C for 12 hours.

The ligation product was transformed into Escherichia coli TG1 strain, and recombinant transformants were obtained by blue-white culture and screening, and plasmid DNA was extracted in small batches (see: "Molecular Cloning", 1989, Science Press), and identified by EcoR V and Xho I digestion. The 230bp fragment was cloned, and it was proved by sequencing that the IGF-I gene had been successfully cloned, and the obtained recombinant plasmid was called pSK-IGF.

3.A3 promoter controls the construction of IGF-I gene

According to the sequence of pigA3GFP (see: Cary, L.C. et al. Transposon mutagenesis ofbaculoviruses: analysis of Trichoplusia ni transposon IFP2 insertions within the FP-locus of nuclear polyhedrosis viruses. Virology, 1989, 172: 156-69), design specific primers A3- 1(tag aat tct gcg cgt tac cat ata tgg tg) and A3-2(ctgata tca gta tta tta aat aag tga caa gta c), using pigA3GFP as a template, the A3 promoter (about 210bp) was amplified by conventional PCR. EcoR I and EcoR V were digested and cloned into the pSK-IGF vector with the same double enzymes to obtain the recombinant pSK-A3-IGF vector.

Design specific primers TPFib-L-3 (ggc tcg agc aaa ttg tgt ttg cgt tag g) and TPFib-L-4 (gcg gta cccact gtc caa tcc acc gtc) according to the GenBank published sequence (GenBank accession number M76430), A fragment of about 290 bp was amplified using the genomic DNA of the silkworm pine variety as a template, which was confirmed to be the polyA signal sequence of the silk fibroin light chain gene by sequencing. The pSK-A3-IGF vector was ligated to obtain the recombinant vector pSK-A3-IGF-polyA.

4. Construction of piggyBAC transposon-based transgenic vector with silk protein light chain enhancer element

According to the sequence of the published silk protein light chain gene (GenBank accession number M76430), the heterosexual primers TPFib-L-5 (cgg ata tct atg ggc tcc agt aac c) and TPFib-L-6 (gcg tcgacg gtc agg tta gat taa cgg g), using silkworm genome DNA as a template, conventional PCR amplification to obtain about 400bp sequence with enhancer function in the first intron of silk fibroin light chain gene, after double digestion with Sal I and EcoR V, Cloned into the PigA3GFP plasmid digested by Sal I and Sma I, a piggyBAC transposon-based transgenic vector with enhancer elements was obtained, named pigA3-fiben.

5. Construction of transgenic vector with enhancer element and A3 promoter to control IGF-I exogenous gene

pSK-A3-IGF-polyA was digested with EcoR I and Kpn I, and the fragment of A3 controlling the IGF-I gene was recovered, cloned into the pigA3-fiben vector digested with EcoR I and Kpn I, and the obtained new vector was named pigA3 -A3-IGF-fiben

6. Construction of the recombinant vector with the IE-1 gene promoter of the silkworm nuclear polyhedrosis virus controlling the neomycin resistance gene neo

Using the DNA of the silkworm nuclear polyhedrosis virus as a template, the IE-1 gene promoter specific primer pair (5′ttc gaa ttc gat ttg cag ttc ggg ac3′, 5′gcg gat atc agt cgt ttg gtt gtt ca3′ ) for PCR amplification, the PCR product was digested with EcoR I and EcoR V, and then cloned into the pBluescript II SK (+) vector to obtain the vector pSK-IE with the IE-1 gene promoter of the silkworm nuclear polyhedrosis virus.

Using pcDNA3.1 vector (product of Invitrogen Company) as template, Neo1 (5′ctg ata tcatga ttg aac aag atg g3′) and Neo3 (5′agc tcg aga att cta gct aga ggt cga c3′) as primers, PCR amplification The coding sequence of the neo gene and its downstream polyA signal sequence (about 1.1kb) were added, digested with EcoR V and Xho I, and then cloned into the pSK-IE vector that had been treated in the same way to obtain the pSK-IE-Neo vector .

7. Construction of recombinant transgenic vector with A3 control IGF-I expression cassette, enhancer element, neomycin resistance gene (neo) and fluorescent protein reporter gene

The pSK-IE-Neo vector was digested with EcoR I, and the fragment IE-Neo with the IE promoter controlling the neomycin resistance gene was recovered, cloned into the same digested pigA3-A3-IGF-fiben plasmid, and the heavy plasmid obtained Named pigA3-A3-IGF-fiben-neo.

8. Screening of Transgenic Cells

Cell transfection was carried out by conventional liposome-mediated method. Take two eppendorf tubes respectively, add 2 μg of the recombinant plasmid pigA3-A3-IGF-fiben-neo to tube A, and 4 μg of the helper plasmid helper (see: TamuraToshiki.et al.Germline transformation of the silkworm Bombyx mori L.using a piggyBac transposon-derived vector.Nature Biotechnology, 2000, 18: 81-84), the total volume was made up to 50 μL with TC-100 medium (FBS Free), mixed well and set aside. Add 10 μL of lipofection reagent (Lipofection) to tube B, make up the total volume to 50 μL with TC-100 medium (FBSFree), mix tubes A and B, and let stand at room temperature for 30 minutes before transfecting Bm-N cells . On the 4th day of transfection, G418 was added to a final concentration of 1000 μg/mL. After one month of selection and culture, G418 was added to a final concentration of 1,500 μg/mL, and the selection was continued for 2 months to obtain cells transfected with the IGF-I gene.

9. Method for cloning transgenic cells expressing IGF-I at a high level

The obtained IGF-I gene transfected cell line was injected into a 96-well plate by extreme dilution, observed with an inverted fluorescence microscope to find a well with only one fluorescent cell, injected into normal Bm-N cells and cultured for 1 week, and added G418 to a final concentration of 1000 μg/ mL, after screening and culturing for 1 month, add G418 to a final concentration of 1,500 μg/mL, and continue screening for 2 months to obtain cloned cell lines. ELISA detection was performed on the cloned cell lines, and a high-level (4 μg/mL) expression of IGF- The cloned cells of I, namely engineered cells.

Example 2: A method for improving the expression of human granulocyte-macrophage colony-stimulating factor in transgenic Spodoptera frugiperda Sf cells (hGM-CSF under the control of the A3 promoter, the first intron fiben of the silk protein light chain, IE- 1 controlled neo), the specific steps include:

1. Preparation of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) gene

Add 100 mg of human T-lymphoid cell line cells, grind with liquid nitrogen, add 1 mL of Trozol RNA extraction solution produced by GIBCO BRL Company, shake gently for 10 minutes, add 500 μL of chloroform, place at room temperature for 10 minutes, and centrifuge at 12,000 rpm for 10 minutes. Take the supernatant, add 2 times the supernatant volume of 100% cold ethanol, mix well, centrifuge at 12,000rpm for 10 minutes, discard the supernatant, add reverse transcriptase and 4dNTPs for reverse transcription, 37°C for 1 hour, and obtain mRNA The synthesized cDNA was reverse transcribed.

The obtained cDNA was added with upstream primers of the hGM-CSF gene (tggatatcACCATGGGCatgtggctgcagagcctgc, atctcgagaagcttatcactcctggactggctccc,) for PCR amplification to obtain a hGM-CSF cDNA fragment with a length of about 450 bp.

2. Construction of pBluescript II SK(+) recombinant plasmid with hGM-CSF gene

The obtained cDNA fragment of hGM-CSF was double-digested with EcoR V and Xho I, and the digested fragment was recovered by conventional methods, and ligated with the pBluescript II SK (+) plasmid (product of Stratagene) double-digested with EcoR V and Xho I. T4 DNA ligase is a product of GIBCO BRL company, and the ligation condition is 16°C for 12 hours.

The ligation product was transformed into Escherichia coli TG1 strain, and recombinant transformants were obtained by blue-white culture and screening, and plasmid DNA was extracted in small batches (see: "Molecular Cloning", 1989, Science Press), and identified by EcoR V and Xho I digestion. The 450bp fragment was cloned, and the hGM-CSF gene was successfully cloned through sequencing, and the obtained recombinant plasmid was called pSK-hGM-CSF.

3. Construction of hGM-CSF gene controlled by A3 promoter

According to the sequence of pigA3GFP (see: Cary, L.C. et al. Transposon mutagenesis ofbaculoviruses: analysis of Trichoplusia ni transposon IFP2 insertions within the FP-locus of nuclear polyhedrosis viruses. Virology, 1989, 172: 156-69), design specific primers A3- 1 (tag aat tct gcg cgt tac cat ata tgg tg, ctg ata tcagta tta tta aat aag tga caa gta c), using pigA3GFP as a template, the A3 promoter (about 210 bp) was amplified by conventional PCR, which was amplified by EcoR I and EcoR V After double digestion, it was cloned into the same double digestion pSK-hGM-CSF vector to obtain the recombinant pSK-A3-hGM-CSF vector.

Design specific primers TPFib-L-3 (ggc tcg agc aaa ttg tgt ttg cgt tag g) and TPFib-L-4 (gcg gta cccact gtc caa tcc acc gtc) according to the GenBank published sequence (GenBank accession number M76430), A fragment of about 290 bp was amplified using the genomic DNA of the silkworm pine variety as a template, which was confirmed to be the polyA signal sequence of the silk fibroin light chain gene by sequencing. The pSK-A3-hGM-CSF vector was ligated to obtain the recombinant vector pSK-A3-hGM-CSF-polyA.

4. Construction of transgenic vector based on piggyBAC transposon with silk protein light chain enhancer element fiben

Same as step 4 in Example 1.

5. Construction of transgenic vector with enhancer element fiben and A3 promoter controlling hGM-CSF exogenous gene

pSK-A3-hGM-CSF-polyA was digested with EcoR I and Kpn I, the fragment of A3 controlling the hGM-CSF gene was recovered, cloned into the pigA3-fiben vector digested with EcoR I and Kpn I, and the new vector obtained was named is pigA3-A3-hGM-CSF-fiben.

6. Construction of the recombinant vector with the IE-1 gene promoter of the silkworm nuclear polyhedrosis virus controlling the neomycin resistance gene neo

Same as step 6 in Example 1.

7. Construction of recombinant transgenic vector with A3 control hGM-CSF expression cassette, enhancer element, neomycin resistance gene (neo) and fluorescent protein reporter gene

The pSK-IE-Neo vector was digested with EcoR I, and the fragment IE-Nco with the IE promoter controlling the neomycin resistance gene was recovered, and cloned into the same digested pigA3-A3-hGM-CSF-fiben plasmid to obtain The heavy plasmid was named pigA3-A3-hGM-CSF-fiben-neo.

8. Screening of Transgenic Cells

Cell transfection was carried out by conventional liposome-mediated method. Take two eppendorf tubes respectively, add 2 μg of recombinant plasmid pigA3-A3-hGM-CSF-fiben-neo to tube A, and 4 μg of helper plasmid helper (see Tamura Toshiki.et al. Germline transformation of the silkworm Bombyxmori L.using a piggyBac transposon- derived vector.Nature Biotechnology, 2000, 18:81-84), the total volume was made up to 50 μL with TC-100 medium (FBS Free), mixed well and set aside. Add 10 μL of lipofection reagent (Lipofection) to tube B, make up the total volume to 50 μL with TC-100 medium (FBSFree), mix tubes A and B, leave at room temperature for 30 minutes, and then transfect the tuberculosis Sf cell. On the 4th day of transfection, G418 was added to a final concentration of 1000 μg/mL. After one month of selection and culture, G418 was added to a final concentration of 1,500 μg/mL, and the selection was continued for 2 months to obtain cells transfected with hGM-CSF gene.

9. Cloning of transgenic cells expressing high levels of hGM-CSF

The hGM-CSF gene transgenic cells obtained were divided into 96-well plates by extreme dilution, observed with an inverted fluorescence microscope to find the wells with only one fluorescent cell, injected into normal sf cells and cultured for 1 week, added G418 to a final concentration of 1000 μg/mL, and screened After culturing for 1 month, add G418 to a final concentration of 1,500 μg/mL, and continue to screen for 2 months to obtain cloned cell lines, and perform ELISA detection on the cloned cell lines to screen out clones expressing hGM-CSF at a high level (5 μg/mL) Cells, namely engineered cells.

Embodiment three: a kind of method (the hGM-CSF under the control of A3 promoter control, the en of hr3, the neo of IE-1 control) that improves transgenic silkworm cell expresses hGM-CSF, specifically comprises the following steps:

1. Preparation of human granulocyte-macrophage colony-stimulating factor gene

Same as step 1 in implementation example two.

2. Construction of pBluescript II SK(+) recombinant plasmid with hGM-CSF gene

Same as step 2 in the implementation example two.

3. Construction of hGM-CSF gene controlled by A3 promoter

Same as step 3 in the implementation example two.

4. Construction of transgenic vector based on piggyBAC transposon with hr3 enhancer element

Primers were designed according to the public sequence of GenBank accession number L33180, using the genomic DNA of silkworm nuclear polyhedrosis virus (BmNPV) as a template, and TPhr3-1 (ctg gta ccg ccg tgc cca gtcacg tgt acg cc) and TPhr3-2 (ctc ccg ggg tga aca gcc cat tcg agg c) as primers, routine PCR amplification, obtained about 740bp sequence with enhancer function in hr3 of the silkworm nucleopolyhedrosis virus (BmNPV) homologous region sequence (homologous region), Kpn I and After double digestion with Sma I, clone into the PigA3GFP plasmid digested with Kpn I and Sma I to obtain a transgenic vector based on piggyBAC transposon with hr3 enhancer element, named pigA3-hr3en.

5. Construction of transgenic vector with enhancer elements hr3 and A3 promoter controlling hGM-CSF exogenous gene

pSK-A3-hGM-CSF-polyA was digested with EcoR I and Kpn I, the fragment of A3 controlling the hGM-CSF gene was recovered, cloned into the pigA3-hr3en vector digested with EcoR I and Kpn I, and the new vector obtained was named pigA3-A3-hGM-CSF-hr3en

6. Construction of the recombinant vector with the IE-1 gene promoter of the silkworm nuclear polyhedrosis virus controlling the neomycin resistance gene neo

Same as step 6 in Example 1.

7. Construction of recombinant transgenic vector with A3 control hGM-CSF expression cassette, enhancer element, neomycin resistance gene (neo) and fluorescent protein reporter gene

The pSK-IE-Neo vector was digested with EcoR I, and the fragment IE-Neo with the IE promoter controlling the neomycin resistance gene was recovered, cloned into the same digested pigA3-A3-hGM-CSF-hr3en plasmid, and the obtained The heavy plasmid was named pigA3-A3-hGM-CSF-hr3en-neo.

8. Screening of Transgenic Cells

Same as Step 8 in Example 2, except that pigA3-A3-hGM-CSF-fiben-neo is replaced by pigA3-A3-hGM-CSF-hr3en-neo, and the cells are Bm-N cells of silkworm.

9. Cloning of transgenic silkworm cells expressing hGM-CSF at a high level

The method is the same as step 9 in the implementation example two. The difference is that transgenic silkworm cells expressing hGM-CSF at a high level (5 μg/mL) were obtained through extreme dilution and actual detection by ELISA.

Embodiment four: a kind of method that improves transgenic silkworm cell to express IGF-1 (IGF-1 under the control of IE promoter, hr3 enhancer, neo controlled by IE-1), specifically comprises the following steps:

1. Preparation of human insulin-like growth factor-I gene

Same as step 1 in Example 1.

2. Construction of pBluescript II SK (+) recombinant plasmid with IGF-I gene

Same as step 2 in Example 1.

3. IE promoter controls the construction of IGF-I gene

According to the published sequence of GenBank accession number L33180, the DNA of the silkworm nuclear polyhedrosis virus was used as a template, and the specific primers of the IE-1 gene promoter (5′ttc gaa ttc gat ttg cag ttc gggac3′, 5′gcg gat atc agt cgt ttg gtt gtt ca3') for PCR amplification, the PCR product was digested with EcoR I and EcoR V, and then cloned into the pSK-IGF vector with the same double enzymes to obtain the recombinant pSK-IE-IGF vector.

According to the public sequence of GenBank accession number M76430, design specific primers TPFib-L-3 (ggc tcg agc aaa ttg tgt ttg cgt tag g) and TPFib-L-4 (gcg gta ccc act gtc caa tccacc gtc) The genomic DNA of the pine variety was used as a template, and a fragment of about 290bp was amplified, which was proved to be the polyA signal sequence of the silk fibroin light chain gene by sequencing. -IGF vector connection to obtain the recombinant vector pSK-IE-IGF-polyA.

4. The construction of the piggyBAC transposon-based transgenic vector carrying the hr3 enhancer element is the same as Step 4 in Example 3.

5. Construction of transgenic vector with enhancer element and IE promoter to control IGF-I exogenous gene

pSK-IE-IGF-polyA was digested with EcoR I and Kpn I, and the fragment of the IE promoter controlling the IGF-I gene was recovered, cloned into the pigA3-hr3en vector digested with EcoR I and Kpn I, and the new vector obtained was named for pigA3-IE-IGF-hr3en

6. Construction of the recombinant vector with the IE-1 gene promoter of the silkworm nuclear polyhedrosis virus controlling the neomycin resistance gene neo

Same as step 6 in Example 1.

7. Construct the recombinant transgenic vector with IE control IGF-I expression cassette, enhancer element, neomycin resistance gene (neo) and fluorescent protein reporter gene

The pSK-IE-Neo vector was digested with EcoR I, and the fragment IE-Neo with the IE promoter controlling the neomycin resistance gene was recovered, and cloned into the same digested pigA3-IE-IGF-hr3en plasmid to obtain a heavy plasmid Named pigA3-IE-IGF-hr3en-neo.

8. Screening of Transgenic Cells

Same as step 8 in Example 1. The difference is that pigA3-IE-IGF-hr3en-neo is used instead of pigA3-A3-IGF-fiben-neo.

9. Cloning of transgenic cells expressing high levels of IGF

Same as step 9 in Example 1.

Embodiment five: a kind of method (IGF-1 under the control of A3 promoter control, silk protein light chain 1st intron fiben, the neo of A3 control) of improving transgenic silkworm cell expressing IGF-1, specifically comprises the following steps:

1. Preparation of human insulin-like growth factor-I gene

Same as step 1 in Example 1.

2-construct pBluescript II SK (+) recombinant plasmid with IGF-I gene

Same as step 2 in Example 1.

3.A3 promoter controls the construction of IGF-I gene

Same as step 3 in Example 1.

4. Construction of piggyBAC transposon-based transgenic vector with silk protein light chain enhancer element

Same as step 4 in Example 1.

5. Construction of transgenic vector with enhancer element and A3 promoter to control IGF-I exogenous gene

Same as step 5 in Example 1.

6. Construction of a recombinant vector with the Bombyx mori nuclear polyhedrosis virus A3 promoter controlling the neomycin resistance gene neo

According to the sequence of pigA3GFP (see: Cary, L.C. et al. Transposon mutagenesis ofbaculoviruses: analysis of Trichoplusia ni transposon IFP2 insertions within the FP-locus of nuclear polyhedrosis viruses. Virology, 1989, 172: 156-69), design specific primers A3- 1 (tag aat tct gcg cgt tac cat ata tgg tg, ctg ata tcagta tta tta aat aag tga caa gta c), using pigA3GFP as a template, the A3 promoter (about 210 bp) was amplified by conventional PCR, which was amplified by EcoR I and EcoR V After double-enzyme digestion, it was cloned into the same double-enzyme-digested pBluescript II SK(+) vector to obtain the vector pSK-A3 with the Bombyx mori nuclear polyhedrosis virus A3 promoter.

Using pcDNA3.1 vector (product of Invitrogen Company) as template, Neo1 (5′ctg ata tcatga ttg aac aag atg g3′) and Neo3 (5′agc tcg aga att cta gct aga ggt cga c3′) as primers, PCR amplification The coding sequence of the neo gene and its downstream polyA signal sequence (about 1.1kb) were added, digested with EcoR V and Xho I, and then cloned into the pSK-A3 vector that had undergone the same treatment to obtain the pSK-A3-Neo vector .

7. Construction of recombinant transgenic vector with A3 control IGF-I expression cassette, enhancer element, neomycin resistance gene (neo) and fluorescent protein reporter gene

The pSK-A3-Neo vector was digested with EcoR I, and the fragment IE-Neo with the A3 promoter controlling the neomycin resistance gene was recovered, cloned into the same digested pigA3-A3-IGF-fiben plasmid, and the heavy plasmid obtained Named pigA3-A3-IGF-fiben-A3neo.

8. Screening of Transgenic Cells

Cell transfection was carried out by conventional liposome-mediated method. Take two eppendorf tubes respectively, add 2 μg of recombinant plasmid pigA3-A3-IGF-fiben-A3neo to tube A, and 4 μg of auxiliary plasmid helper (see Tamura Toshiki.et al. Germline transformation of the silkworm Bombyxmori L.using a piggyBac transposon-derived vector .Nature Biotechnology, 2000, 18:81-84), make up the total volume to 50 μL with TC-100 medium (FBS Free), mix well and set aside. Add 10 μL of lipofection reagent (Lipofection) to tube B, make up the total volume to 50 μL with TC-100 medium (FBSFree), mix tubes A and B, and let stand at room temperature for 30 minutes before transfecting Bm-N cells . On the 4th day of transfection, G418 was added to a final concentration of 1000 μg/mL. After one month of selection and culture, G418 was added to a final concentration of 1,500 μg/mL, and the selection was continued for 2 months to obtain the transfected IGF-I cell line.

9. Cloning method of transgenic cells expressing high level of IGF-1

The method is the same as step 9 in the implementation example one.

Claims (5)

1. method that improves transgenic insect cell expression exogenous gene level comprises following concrete steps:

(1) makes up the carrier that has active promotor X control foreign gene in the insect cell;

(2) make up the carrier pigA3-en that has reporter gene that has enhancer element en based on the piggyBAC transposon;

(3) double digestion step (1) gained carrier reclaims the fragment that promotor X controls foreign gene, and the clone advances the pigA3-en of same double digestion, must have the carrier of enhanser en and promotor X control foreign gene;

(4) make up the recombinant vectors that has active promotor Y control neomycin resistance gene neo in the insect cell;

(5) enzyme is cut step (4) gained carrier, reclaim the fragment Y-Neo of promotor Y control neomycin resistance gene, the clone advances step (3) the gained carrier that same enzyme is cut, and obtains having the reorganization transgene carrier of promotor X control exogenous gene expression box, enhancer element, promotor Y control neomycin resistance gene, fluorescent protein report gene;

(6) step (5) gained reorganization transgene carrier is mixed with the helper plasmid of expressing transposase, transfection insect cell system is by the segmentation screening acquisition transgenic insect cell of G418.

2. a kind of method that improves transgenic insect cell expression exogenous gene level according to claim 1 is characterized in that: described insect cell is a bombyx mori cell.

3. a kind of method that improves transgenic insect cell expression exogenous gene level according to claim 1 is characterized in that: described enhancer element is selected from a kind of in the homologous region of silk fibroin protein light chain gene the 1st intron sequences or baculovirus.

4. a kind of method that improves transgenic insect cell expression exogenous gene level according to claim 1, it is characterized in that: have the utmost point early gene IE-1 promotor that active promotor is selected from baculovirus in the described insect cell, also can be in the CMV promotor of silkworm Actin muscle A3 promotor, cytomegalovirus, the insect heat shock protein promotor a kind of.

5. a kind of method that improves transgenic insect cell expression exogenous gene level according to claim 1, it is characterized in that: the transgenic insect cell of step (6) gained is passed through the cell clone technology, in conjunction with the actual detected of exogenous gene expression level, obtain the engineering cell of expression alien gene.