CN1654647A - Method for producing recombinant human lactoferrin in silkworm body by gene engineering technology - Google Patents

Abstract

The present invention discloses gene engineering technology method of producing human lactoferritin (LF) in silkworm body. The hybrid baculovirus HyNPV the present inventor constitutes with AcNPV and BmNPV is used as vector in constituting the recombinant virus containing human LF gene. Using silkworm as the recombinant virus host in expressing and producing recombinant human LF has simple operation, stable process and low cost. Most of the recombinant human LF expressed in silkworm is secreted in silkworm haemolymph and this is favorable to the fast extraction of recombinant human LF. The recombinant human LF is purified in affinity chromatography process and has yield as high as 0.4 mg each silkworm. The present invention has wide application foreground in medicine and treatment.

Description

Utilize genetic engineering technique in the silkworm body, to produce the method for restructuring lactoferrin

Technical field

The present invention relates to genetic engineering technique, baculovirus gene expression system, protein purification, relate in particular to a kind of genetic engineering technique method that great expression is produced reorganization LF in the silkworm body of utilizing.

Background technology

Human lactoferrin Lactoferrin (being abbreviated as LF) has immunocompetent protein by human body mammary gland secretion a kind of, it is made up of 711 amino acid, the about 78KDa of molecular weight, be iron associativity glycoprotein important during the people suckles, just Ruzhong LF content is about 5-7mg/ml, at present confirmed that LF is relevant with people's immunity function, also played an important role at aspects such as absorption that promotes iron and antisepsis and anti-inflammations.Therefore, the metabolism that utilizes LF to have promotion iron is regulated and is antimicrobial, comprise premium propertiess such as bacterium, fungi and virus, the U.S. has developed LF and has infected disease and the full person of complex immunity as functional food additives and as alimentary canal, as acquired immune deficiency syndrome (AIDS), accepted the pharmaceutical preparations of the treatments such as cancer patients of chemotherapy, radiotherapy.Therefore, people LF has broad application prospects.Cost an arm and a leg but LF is proteic in the market, though in intestinal bacteria, can produce with engineered method, but LF need make this process efficiency very low and uneconomical to its folding again its biological activity of recovering with the formal representation of inclusion body in intestinal bacteria.In order to overcome this problem, other people once tried to express production with the mammalian cell of yeast and adenovirus infection, but zymic plasmid transformant instability and Mammals culturing cell production cost is too high.Therefore be necessary to set up the method for a kind of production LF of efficient economy.

Baculovirus expression system is one of the most effective now eukaryotic gene expression system; and " silkworm-recombinant baculovirus expression system " is owing to can utilize the specialty economies insect-silkworm of China's number of animals raised maximum to make to express carrier; silkworm has characteristics such as individual big, easy raising; therefore have low, the advantage such as be produced on a large scale of production cost, be suitable for the requirement of bio-pharmaceuticals industrialization level.The applicant has made up a good expression system HyNPV (patent No. ZL01125605.2) who is applicable to silkworm before this, and it has the outstanding advantage in the host territory of expansion.Like this, can be in the host cell Sf21 of AcNPV construction of recombinant virus, low-cost in silkworm larva then, produce recombinant protein efficiently.

Summary of the invention

The object of the present invention is to provide a kind of recombinant human LF silkworm vivo gene engineering produce method.The hybridization baculovirus HyNPV that utilizes AcNPV that the applicant makes up and BmNPV is as the carrier (patent No.: ZL01125605.2) made up the recombinant virus that contains people LF gene, and utilized silkworm as the host, expressed production recombinant human LF.

In order to achieve the above object, its step of the technical solution used in the present invention is as follows:

1, the recombinant human LF method of producing in silkworm vivo gene engineering:

(1) clone of LF gene: with people's mammary tissue cDNA is template, and the pcr gene amplification technique obtains people LF gene, and design of primers is as follows: forward primer: 5 '- GGATCCATGAAACTTGTCTTCCTC-3 ' contains the BamHI restriction enzyme site; Reverse primer: 5 '- GGATCCTTACTTCCTGAGGAATTC-3 ' contains the BamHI restriction enzyme site.The cycle number of PCR reaction, the design of temperature and time are as follows: a circulation, 94 ℃ of template sex change 50 seconds; 30 circulations were annealed 30 seconds for 55 ℃, and 70 ℃ were extended 2 minutes; Last 1 circulation, 70 ℃ were extended 10 minutes; Utilize 1% agarose gel electrophoresis to carry out the PCR product analysis, and with PCR purification kit purifying, obtaining total length is the LF gene of 2136bp, subsequently the LF gene clone is gone into 3.9kb carrier pCR2.1, utilize dideoxy chain termination on the nucleotide sequencing instrument, to check order and confirm clone LF gene order exactness;

(2) contain the structure of the recombinant baculovirus of people LF gene: pCR2.1-LF obtains the LF gene fragment with restriction enzyme BamHI digestion, is cloned into baculovirus transfer vector pBlueBacHisa then and obtains recombinant chou pBlueBacHisa-LF; The hybridization baculovirus HyNPV that utilizes AcNPV that the applicant makes up and BmNPV then is as the carrier (patent No.: ZL01125605.2), obtain to contain the recombinant virus of people LF gene by cotransfection.The method of cotransfection is: 1 μ g pBlueBacHisa-LF DNA and 15 μ g HyNPVDNA mix, and add 14 μ l liposome lipofectin, add sterile purified water at last to final volume 40 μ l; Sf 21 culturing cells with this mixed solution cotransfection logarithmic phase after room temperature is cultivated 15 minutes.Earlier after 24 hours, change the fresh culture that contains 10% foetal calf serum into, cultivated 5 days for 27 ℃, collect the substratum supernatant, be used for recombinant celo virus as virus stock solution used with the TC-100 culture medium culturing of serum-free; Recombinant virus is taken turns plaque select by 3, finally obtains the viral solution of high density purifying, and concentration is 10 ⁸Pfu/ml, the applicant is with this recombinant virus called after HyNPV-LF;

(3) silkworm expression in vivo and reorganization LF purifying: use silkworm larva to play silkworm 5 ages, inoculate above-mentioned recombinant virus and infect expression; Before the inoculation, larva is immersed in the ice-water bath anesthesia 10 minutes, injection viral suspension concentration is 10 ⁶Pfu/ml adopts hypodermic method, and every silkworm is injected 20 μ l, injects and gives mulberry after 1 hour, raises down at 23-25 ℃; In metainfective three days, can't see tangible symptom, by the 4th day, larva began appetite and weakens, and presented infection symptoms gradually; Infected ground, back five days or the 6th day, the larva of infection is most of dead, before this collector's silkworm larva.With the hemolymph of larva parent material, use following method to collect hemolymph: above the 15ml collection tube, larva to back side bending, with a hand fixedly head and the afterbody of larva, to be allowed outside belly and abdominal foot stretch to as recombinant human LF purifying.No. 25 punctures abdominal foots with the Parafilm sealing are collected blood, allow blood directly drip in the collection tube, in order to prevent the blood oxidation blackening, add the 10mmol/L dithiothreitol (DTT) DTT of 1/10 volume in advance, and making its final concentration is 1mmol/L;

Because recombinant protein N-end has 6 * His tail, can carry out protein purification under non-sex change condition with the Ni-NTA affinity column; The hemolymph of collecting from the larva that infects is with non-sex change binding buffer liquid, Na ₃P0 ₄50mmol/L, NaCl 0.5mol/L, pH 8.0, dilute; Sample is incorporated into the Ni-NTA post subsequently, at last, the recombinant protein non-sex change elution buffer wash-out that contains the 250mmol/L imidazoles, by identifying recombinant protein behind the SDS-PAGE coomassie brilliant blue staining, the result shows that the reorganization LF of acquisition is very single, and the output that obtains from every boss silkworm is up to 400 μ g.

The present invention compares with background technology, and the beneficial effect that has is:

The present invention has solved LF preferably needs in complicated renaturation manipulation, the yeast expression plasmid transformant unstable and in the too high shortcoming of Mammals culturing cell production cost at expression in escherichia coli, and silkworm larva express recombinant people LF has the advantage that cost is low, output is high, biological activity is strong.The method that this patent is set up can mass production recombinant human LF, has broad application prospects in the medical science and the clinical treatment field in future.

Embodiment

1, research material: genetically engineered operational tool enzyme and pcr amplification test kit are purchased in precious Takara biotechnology company limited (Dalian).People's mammary tissue cDNA is available from Clotech company, and TA clone test kit, baculovirus transfer vector pBlueBacHisa, liposome lipofectin are Invitrogen company product.Dna sequencing kit is purchased the Biosystems in PE Applied.The substratum TC-100 of foetal calf serum FCS, insect cell line Sf21 is the product of GibcoBRL.Spodoptera frugiperda cell is that the substratum ESF921 of Tn-5 purchases the systems in Expression.The greedy frugiperda cell Sf21 in meadow uses the TC-100 substratum that adds 10% (v/v) FCS and 0.26% bacto-tryptone 27 ℃ of cultivations.Tn-5 utilizes the ESF921 of serum-free to cultivate based on cultivating in 27 ℃ of bottles.Silkworm hybrid is used in this test, and the silkworm larva mulberry leaf are raised, and temperature is 23～25 ℃.

2, workflow:

The method that recombinant human LF produces in silkworm vivo gene engineering is characterized in that:

(1) clone of LF gene: with people's mammary tissue cDNA is template, and the pcr gene amplification technique obtains people LF gene, and design of primers is as follows: forward primer: 5 '- GGATCCATGAAACTTGTCTTCCTC-3 ' contains the BamHI restriction enzyme site; Reverse primer: 5 '- GGATCCTTACTTCCTGAGGAATTC-3 ' contains the BamHI restriction enzyme site; The cycle number of PCR reaction, the design of temperature and time are as follows: a circulation, 94 ℃ of template sex change 50 seconds; 30 circulations were annealed 30 seconds for 55 ℃, and 70 ℃ were extended 2 minutes; Last 1 circulation, 70 ℃ were extended 10 minutes; Utilize 1% agarose gel electrophoresis to carry out the PCR product analysis, and with PCR purification kit purifying, obtaining total length is the LF gene of 2136bp, subsequently the LF gene clone is gone into 3.9kb carrier pCR2.1, utilize dideoxy chain termination on the nucleotide sequencing instrument, to check order and confirm clone LF gene order exactness;

(2) contain the structure of the recombinant baculovirus of people LF gene: pCR2.1-LF obtains the LF gene fragment with restriction enzyme BamHI digestion, is cloned into baculovirus transfer vector pBlueBacHisa then and obtains recombinant chou pBlueBacHisa-LF; In insect cell, produce recombinant virus by cotransfection with hybridization baculovirus HyNPVDNA homologous recombination, the method of cotransfection is: 1 μ g pBlueBacHisa-LF DNA and 15 μ g HyNPV DNA mix, and add 14 μ l liposome lipofectin, add sterile purified water at last to final volume 40 μ l; Sf 21 culturing cells with this mixed solution cotransfection logarithmic phase after room temperature is cultivated 15 minutes.Earlier after 24 hours, change the fresh culture that contains 10% foetal calf serum into, cultivated 5 days for 27 ℃, collect the substratum supernatant, be used for recombinant celo virus as virus stock solution used with the TC-100 culture medium culturing of serum-free; Recombinant virus is taken turns plaque select by 3, finally obtains the viral solution of high density purifying, and concentration is 10 ⁸Pfu/ml is with this recombinant virus called after HyNPV-LF;

(3) silkworm expression in vivo and reorganization LF purifying: use silkworm larva to play silkworm 5 ages, inoculate above-mentioned recombinant virus and infect expression; Before the inoculation, larva is immersed in the ice-water bath anesthesia 10 minutes, injection viral suspension concentration is 10 ⁶Pfu/ml adopts hypodermic method, and every silkworm is injected 20 μ l, injects and gives mulberry after 1 hour, raises down at 23-25 ℃; In metainfective three days, can't see tangible symptom, by the 4th day, larva began appetite and weakens, and presented infection symptoms gradually; Infected ground, back five days or the 6th day, the larva of infection is most of dead, before this collector's silkworm larva.With the hemolymph of larva parent material, use following method to collect hemolymph: above the 15ml collection tube, larva to back side bending, with a hand fixedly head and the afterbody of larva, to be allowed outside belly and abdominal foot stretch to as recombinant human LF purifying.No. 25 punctures abdominal foots with the Parafilm sealing are collected blood, allow blood directly drip in the collection tube, in order to prevent the blood oxidation blackening, add the 10mmol/L dithiothreitol (DTT) DTT of 1/10 volume in advance, and making its final concentration is 1mmol/L;

Because recombinant protein N-end has 6 * His tail, can carry out protein purification under non-sex change condition with the Ni-NTA affinity column; The hemolymph of collecting from the larva that infects is with non-sex change binding buffer liquid, Na ₃PO ₄50mmol/L, NaCl 0.5mol/L, pH 8.0, dilute; Sample is incorporated into the Ni-NTA post subsequently, at last, the recombinant protein non-sex change elution buffer wash-out that contains the 250mmol/L imidazoles, by identifying recombinant protein behind the SDS-PAGE coomassie brilliant blue staining, the result shows that the reorganization LF of acquisition is very single, and the output that obtains from every boss silkworm is up to 400 μ g.

Claims (1)

1, utilize genetic engineering technique in the silkworm body, to produce the method for restructuring lactoferrin, it is characterized in that:

(1) clone of LF gene: with people's mammary tissue cDNA is template, and the pcr gene amplification technique obtains people LF gene, and design of primers is as follows: forward primer: 5 '- GGATCCATGAAACTTGTCTTCCTC-3 ' contains the BamHI restriction enzyme site; Reverse primer: 5 '- GGATCCTTACTTCCTGAGGAATTC-3 ' contains the BamHI restriction enzyme site; The cycle number of PCR reaction, the design of temperature and time are as follows: a circulation, 94 ℃ of template sex change 50 seconds; 30 circulations were annealed 30 seconds for 55 ℃, and 70 ℃ were extended 2 minutes; Last 1 circulation, 70 ℃ were extended 10 minutes; Utilize 1% agarose gel electrophoresis to carry out the PCR product analysis, and with PCR purification kit purifying, obtaining total length is the LF gene of 2136bp, subsequently the LF gene clone is gone into 3.9kb carrier pCR2.1, utilize dideoxy chain termination on the nucleotide sequencing instrument, to check order and confirm clone LF gene order exactness;

(2) contain the structure of the recombinant baculovirus of people LF gene: pCR2.1-LF obtains the LF gene fragment with restriction enzyme BamHI digestion, is cloned into baculovirus transfer vector pBlueBacHisa then and obtains recombinant chou pBlueBacHisa-LF; With the hybridization baculovirus HyNPV DNA that makes up homologous recombination taking place by cotransfection produces recombinant virus in insect cell, the method of cotransfection is: 1 μ gpBlueBacHisa-LF DNA and 15 μ g HyNPV DNA mix, and add 14 μ l liposome lipofectin, add sterile purified water at last to final volume 40 μ l; Sf21 culturing cell with this mixed solution cotransfection logarithmic phase after room temperature is cultivated 15 minutes, use the TC-100 culture medium culturing of serum-free after 24 hours earlier, change the fresh culture that contains 10% foetal calf serum into, cultivated 5 days for 27 ℃, collect the substratum supernatant as virus stock solution used, be used for recombinant celo virus; Recombinant virus is taken turns plaque select by 3, finally obtains the viral solution of high density purifying, and concentration is 10 ⁸Pfu/ml is with this recombinant virus called after HyNPV-LF;

(3) silkworm expression in vivo and reorganization LF purifying: use silkworm larva to play silkworm 5 ages, inoculate above-mentioned recombinant virus HyNPV-LF and infect expression; Before the inoculation, larva is immersed in the ice-water bath anesthesia 10 minutes, injection viral suspension concentration is 10 ⁶Pfu/ml adopts hypodermic method, and every silkworm is injected 20 μ l, injects and gives mulberry after 1 hour, raises down at 23-25 ℃; In metainfective three days, can't see tangible symptom, by the 4th day, larva began appetite and weakens, and presented infection symptoms gradually; Infected ground, back five days or the 6th day, the larva of infection is most of dead, before this collector's silkworm larva; With the hemolymph of larva parent material, use following method to collect hemolymph: above the 15ml collection tube, larva to back side bending, with a hand fixedly head and the afterbody of larva, to be allowed outside belly and abdominal foot stretch to as recombinant human LF purifying; No. 25 punctures abdominal foots with the Parafilm sealing are collected blood, allow blood directly drip in the collection tube, in order to prevent the blood oxidation blackening, add the 10mmol/L dithiothreitol (DTT) DTT of 1/10 volume in advance, and making its final concentration is 1mmol/L;

Because recombinant protein N-end has 6 * His tail, can carry out protein purification under non-sex change condition with the Ni-NTA affinity column; The hemolymph of collecting from the larva that infects is with non-sex change binding buffer liquid, Na ₃PO ₄50mmol/L, NaCl 0.5mol/L, pH8.0 dilutes; Sample is incorporated into the Ni-NTA post subsequently, at last, recombinant protein is with the non-sex change elution buffer wash-out that contains the 250mmol/L imidazoles, by identifying recombinant protein behind the SDS-PAGE coomassie brilliant blue staining, the result shows that the reorganization LF of acquisition is very single, and the output that obtains from every boss silkworm is 400 μ g.