CN101629186A - Method for producing recombinant scorpion toxin protein by adopting silkworm as parasitifer - Google Patents

Abstract

The invention discloses a method for producing recombinant scorpion toxin protein using silkworm as a host, which mainly includes the following steps: using scorpion cDNA as a template, using 5'-ACGTAGATCTATGGATGGATATATAAG-3' containing a BglII restriction site as a forward primer, and using The 5′-ACGTAGATCT TTACTTTTTTTCCAC-3′ containing the BglII restriction site was used as the reverse primer to carry out thirty-two cycles of PCR gene amplification reaction to obtain the scorpion toxin gene; pCR2.1-scorpion toxin was digested with restriction endonuclease BglII The scorpion toxin gene fragment was obtained, and then cloned into the baculovirus transfer vector pAcGP67b to obtain the recombinant pAcGP67b-scorpion toxin gene, and the recombinant virus was produced by co-transfection and homologous recombination with the silkworm baculovirus BmNPV DNA in insect cells; expression and Purification of recombinant scorpion toxin. In the present invention, most of the recombinant scorpion toxin protein expressed by silkworm larvae remains in the fat body of silkworm, and the scorpion toxin is purified from the fat body by affinity chromatography. The recombinant scorpion toxin has obvious biological toxicity, and is suitable for the industrialization level of biopharmaceuticals. Requirements, it has broad application prospects in the field of medicine and clinical treatment in the future.

Description

Method for producing recombinant scorpion toxin protein using silkworm as host

technical field

The invention relates to gene recombination technology, baculovirus gene expression system, protein purification and activity analysis, in particular to a method for producing recombinant scorpion toxin protein using silkworm as a host.

Background technique

Scorpion toxin protein BmK is a small molecular weight protein synthesized and secreted by scorpions. The gene length is 198 nucleotides, encoding 65 amino acids, pI is 6.3, and the predicted molecular weight is about 8KDa, which has strong toxicity. Scorpion toxin has anti-viral, anti-bacterial, anti-tumor, anti-inflammatory, dredging collaterals, dispelling rheumatism, antispasmodic, tranquilizing, pain-relieving and other functions. Therefore, scorpion toxin has good application value and shows a good application prospect. However, the price of scorpion toxin protein on the market is relatively expensive. Although it can be produced in Escherichia coli by genetic engineering, the scorpion toxin is expressed in the form of inclusion bodies in E. coli, and it needs to be refolded to restore its biological activity. The extra handling makes the process very inefficient and uneconomical. To overcome this problem, others have attempted expression production in yeast and adenovirus-infected mammalian cells. However, plasmid transformants of yeast are unstable and expensive to produce in cultured mammalian cells. Therefore, it is necessary to establish an efficient and economical method for producing scorpion toxin.

The baculovirus expression system is one of the most effective eukaryotic gene expression systems today. At present, the system mainly includes Autographa californica nuclear polyhedrosis virus (AcNPV) which is widely used in Europe and the United States, and AcNPV for short. The expression systems of Bombyx mori nuclear polyhedrosis virus (BmNPV) represented by China and Japan are two types. Both have their own advantages, but the host range of the virus is different, and the host range of AcNPV is wider.

Contents of the invention

The purpose of the present invention is to provide a method for producing recombinant scorpion toxin protein using silkworm as a host.

The inventive idea of the present invention is to use BmNPV to construct a recombinant virus containing scorpion toxin gene, and use silkworm as a host to express and produce biologically active recombinant scorpion toxin using the "bombyx mori-recombinant baculovirus expression system".

In order to achieve the above object, the technical solution adopted in the present invention is that the method for producing recombinant scorpion toxin protein with silkworm as host mainly includes the following steps:

(1) Use scorpion cDNA as template, 5′-ACGT AGATCT ATGGATGGATATATAAG-3′ containing BglII restriction site as forward primer, and 5′-ACGT AGATCT TTACTTTTTTCAC-3′ containing BglII restriction site as reverse primer Thirty-two cycles of PCR gene amplification reactions are carried out to the primers to obtain the scorpion toxin gene. The first cycle of the PCR gene amplification reaction is to denature the template at 94° C. for 50 seconds; the second to the thirty-first cycles It is first annealed at 55°C for 30 seconds, and then extended at 70°C for 30 seconds; the thirty-second cycle is extended at 70°C for 7 minutes;

Purify the scorpion toxin gene obtained from the PCR gene amplification reaction, and clone the scorpion toxin gene into the 3.9kb vector pCR2.1 to obtain pCR2.1-scorpion toxin, and confirm the pCR2.1-scorpion toxin by sequencing on a nucleotide sequencer The correctness of the toxin gene sequence;

(2) digest pCR2.1-scorpion toxin with restriction endonuclease BglII to obtain the scorpion toxin gene fragment, then clone the scorpion toxin gene fragment into the baculovirus transfer vector pAcGP67b to obtain the recombinant pAcGP67b-scorpion toxin gene;

Homologous recombination with silkworm baculovirus BmNPV DNA in silkworm cultured cells to produce recombinant virus by co-transfection, the co-transfection method is: first mix 1 μg pAcGP67b-scorpion toxin gene DNA and 15 μg BmNPV DNA, then add 14 μl lipid Plastid lipofectin, and then add sterilized distilled water to a final volume of 40 μl, and finally incubate the mixture at room temperature for 15 minutes before co-transfecting Sf21 cultured cells in logarithmic growth phase;

The above-mentioned recombinant virus produced by co-transfection was first cultured with serum-free TC-100 medium for 24 hours, and then replaced with fresh culture medium containing 10% fetal calf serum. After 5 days of culture at 27°C, the medium supernatant was collected as The virus stock solution is used to screen the recombinant virus; the recombinant virus passes through 3 rounds of plaque screening, and finally obtains a high-concentration purified recombinant BmNPV-scorpion toxin virus solution, and the concentration of the BmNPV-scorpion toxin virus solution is 10 ⁸ pfu/ml;

(3) First, the 5th instar silkworm larvae were immersed in an ice water bath for anesthesia, and then the recombinant BmNPV-scorpion toxin virus suspension with a concentration of 10 ⁶ pfu/ml was injected into the silkworm larvae for infection and expression by subcutaneous injection. Bombyx mori were injected with 20 μl; 1 hour after the injection, silkworm larvae were fed with mulberry and raised at 23-25°C;

Collect silkworm larvae within 72-96 hours after infection, dissect the fat body of silkworm after infection, and use the fat body as the starting material for purifying recombinant scorpion toxin; dissolve the fat body in a pre-cooled pH of 7.6 after homogenizing 20mM Tris-HCl buffer solution, after high-speed centrifugation, put the supernatant directly on the Ni-NAT affinity chromatography column to bind the recombinant scorpion toxin protein to the chromatography column; then use 20mM Tris-HCl buffer with pH 7.6 Impurities were removed by elution with 250 mM imidazole buffer, and the combined recombinant scorpion toxin protein was eluted in one step with 250 mM imidazole buffer.

Compared with the background technology, the present invention has the beneficial effects of:

(1) The recombinant scorpion toxin expressed by silkworm larvae of the present invention has the advantages of low cost, high yield, and strong biological activity, and overcomes the complex renaturation operation and plasmid transformation in yeast expression required for the expression of scorpion toxin in Escherichia coli in the prior art The shortcomings of unstable body and high cost of production in mammalian cultured cells;

(2) Most of the recombinant scorpion toxin protein expressed by the silkworm larva of the present invention remains in the fat body of the silkworm, and the scorpion toxin is purified from the fat body by affinity chromatography, and the yield of each silkworm is as high as 600-700 μg; and the activity analysis shows that, The recombinant scorpion toxin obtained by the present invention has obvious biological toxicity;

(3) The "bombyx mori-recombinant baculovirus expression system" of the present invention can use the characteristic economic insect with the largest feeding amount in my country-bombyx mori as an expression vector, and silkworm has the characteristics of large individual and easy breeding, so it has low production cost, With the advantages of large-scale production, it is suitable for the requirements of the industrialization level of biopharmaceuticals, and has broad application prospects in the fields of medicine and clinical treatment in the future.

Description of drawings

Fig. 1 is the map of the baculovirus transfer vector pAcGP67b used in the present invention;

Fig. 2 is the photo before and after the recombinant scorpion toxin virus of silkworm larva infection among the present invention: the photo before the top left of Fig. 2 is the photo of silkworm infection, and the bottom left is the photo after silkworm infection; The right side is the photo of recombinant scorpion toxin protein purification, wherein, 1 2 is the reference molecular weight (14.3 KDa and 6.0 KDa molecular weight from top to bottom), 2 is the sample as the control, and 3 is the sample purified from infected larvae.

Detailed ways

1. Research materials: genetic engineering operation tools, enzymes and PCR amplification kits were purchased from Shanghai Sangon Bioengineering Technology Service Company in my country. The scorpion cDNA was purchased from Clotech. The TA cloning kit, the baculovirus transfer vector pAcGP67b shown in Figure 1, and the liposome lipofectin were all products of Invitrogen. Products for Ni-NTA affinity chromatography were purchased from Wakochemical Co., Ltd., Japan. DNA sequencing kits were purchased from PE Applied Biosystems. Fetal calf serum FCS, medium TC-100 for insect cell line Sf21 and medium 199 for culturing umbilical vein endothelial cells HUVEC are all products of GibcoBRL. The medium ESF921 of the Fall Armyworm cell line Tn-5 was purchased from Expression systems. Umbilical vein endothelial cells HUVEC, SCS and ECGS are all products of Technoclone GmbH. Spodoptera frugiperda cells Sf21 were cultured at 27°C in TC-100 medium supplemented with 10% (v/v) FCS and 0.26% bacto-tryptone. Tn-5 was cultured using serum-free ESF921 based on flask culture at 27°C. In this experiment, silkworm hybrids were used, and silkworm larvae were raised on mulberry leaves at a temperature of 23-25°C.

2. Production steps:

The method of utilizing silkworm as host to produce recombinant scorpion toxin mainly includes the following steps: (1) cloning of scorpion toxin gene: using East Asian pincer scorpion (Buthus martensii Karsch) cDNA as template, PCR gene amplification technique obtains scorpion toxin gene, and primers are designed as follows : Forward primer: 5′-ACGT AGATCT ATGGATGGATATATAAG-3′ contains a BglII restriction site; reverse primer: 5′-ACGT AGATCT TTACTTTTTTTCCAC-3′ contains a BglII restriction site. The cycle number, temperature and time of the PCR reaction were designed as follows: the first cycle, template denaturation at 94°C for 50 seconds; the second to the thirty-first cycle, annealing at 55°C for 30 seconds, and extension at 70°C for 30 seconds; 1 cycle, 70°C extension for 7 minutes; use 1% agarose gel electrophoresis for PCR product analysis, and use Qiagene’s PCR purification kit to purify, and then clone the target PCR product into the 3.9kb vector pCR2.1 to obtain pCR2.1-scorpion Toxin, use the dideoxy chain termination method to sequence on a nucleotide sequencer to confirm the cloned scorpion toxin gene, that is, to obtain the correctness of the sequence of the pCR2.1-scorpion toxin gene;

(2) Construction of recombinant baculovirus containing scorpion toxin gene: pCR2.1-scorpion toxin was digested with restriction endonuclease BglII to obtain scorpion toxin gene fragment, and then cloned into baculovirus transfer vector pAcGP67b to obtain recombinant pAcGP67b-scorpion Toxin gene; through co-transfection in insect cells and homologous recombination of silkworm baculovirus BmNPV DNA to produce recombinant virus, the co-transfection method is: 1 μg pAcGP67b-scorpion toxin gene DNA and 15 μg BmNPV DNA are mixed, and 14 μl lipid is added Finally, add sterilized distilled water to a final volume of 40 μl; incubate the mixture at room temperature for 15 minutes and co-transfect Sf 21 cultured cells in logarithmic growth phase. After culturing with serum-free TC-100 medium for 24 hours, replace it with fresh medium containing 10% fetal bovine serum, culture at 27°C for 5 days, collect the medium supernatant as the virus stock solution, and use it to screen recombinant viruses; The virus passes through three rounds of plaque screening to finally obtain a high-concentration purified recombinant virus solution with a concentration of 10 ⁸ pfu/ml. The present invention names this recombinant virus BmNPV-scorpion toxin;

(3) In vivo expression and purification of recombinant scorpion toxin in silkworm: Bombyx mori larvae from the 5th instar are used to inoculate the above-mentioned recombinant virus for infection and expression; before inoculation, the larvae are immersed in ice water bath for short-term anesthesia, and then subcutaneous injection is used to inject the recombinant Virus suspension with a concentration of 10 ⁶ pfu/ml, inject 20 μl per silkworm, feed mulberry 1 hour after injection, and raise them at 23-25°C; collect silkworm larvae within 72-96 hours after infection, and dissect the fat of infected silkworms and use this as the starting material for the purification of recombinant scorpion toxin, and silkworm before infection as a control; homogenate the fat body, dissolve it in pre-cooled pH 7.6, 20mM Tris-HCl buffer, and centrifuge at high speed The supernatant is directly applied to the Ni-NAT affinity chromatography column. Due to the strong affinity between the scorpion toxin protein and Ni-NAT, the scorpion toxin protein is bound to the chromatography column, and then eluted with the same buffer containing the above to remove impurities. Protein, and finally the bound recombinant scorpion toxin protein was eluted in one step with 250mM imidazole buffer. The recombinant scorpion toxin protein was identified by SDS-PAGE electrophoresis analysis. Fig. 2 shows the photos before and after the silkworm larva infection of the recombinant scorpion toxin virus in the present invention: the upper left of Fig. 2 is the photo before the silkworm infection, and the lower left is the photo after the silkworm infection; the right side of Fig. 2 is the recombinant scorpion toxin protein purification Photos, wherein, 1 is the molecular weight as a reference, the molecular weights from top to bottom are 14.3KDa and 6.0KDa respectively, 2 is a sample as a control, and 3 is a sample purified from infected larvae. As can be seen from Figure 2, the recombinant scorpion toxin obtained in the present invention has a high purity.

Analysis and identification of biological activity of recombinant scorpion toxin protein: the purified recombinant scorpion toxin protein was dissolved in phosphate buffer by oral feeding method, and the concentrations were prepared to be 50 μg/ml, 100 μg/ml, 200 μg/ml, 500 μg/ml feeding liquid, spray these four feeding liquids on mulberry leaves, feed a group of 4th instar silkworm larvae according to each feeding liquid, each group has 30 larvae. After 72 hours, the larvae were observed for poisoning and death. The results show that the larvae fed by different concentrations of recombinant scorpion toxin protein solution are compared with the control sample shown in Figure 2, and all show a certain degree of poisoning symptoms. As the concentration increases, the degree of poisoning deepens. 12% and 38% of the larvae died due to poisoning in two concentrations of 500μg/ml respectively. The results of this experiment indicated that the recombinant scorpion toxin protein expressed in silkworm larvae had biological toxicity.

Scorpion toxin gene BmK nucleotide sequence list

<110> Zhejiang University

<120> Method for producing recombinant scorpion toxin protein using silkworm as host

<160>1

<170>PatentIn version 3.1

<210>1

<211>198

<212>DNA

<213> East Asian pincer scorpion (Buthus martensii Karsch)

<400>1

atggatggat atataagagg aagtaacgga tgcaaggttt catgcttatg gggaaatgaa 60

ggttgcaata aagaatgcgg agcgtacggt gcctcttatg gttattgctg gacctgggga 120

cttgcatgct ggtgtgaagg ccttcctgat gataagacat ggaaatctga aagtaataca 180

tgcggtggaa aaaagtaa 198

Claims (1)

1. produce reorganization scorpion toxin proteic method with silkworm as the host for one kind, it is characterized in that comprising the steps:

(1) with scorpion cDNA be template, to contain the BglII restriction enzyme site

5 '-ACGT AGATCTATGGATGGATATATAAG-3 ' be forward primer, with contain 5 of BglII restriction enzyme site '-ACGT AGATCTTTACTTTTTTCCAC-3 ' obtains the scorpion toxin gene for reverse primer carries out 32 round-robin pcr gene amplified reactions, and first circulation of described pcr gene amplified reaction is 94 ℃ of template sex change 50 seconds; Second is that elder generation annealed 30 seconds at 55 ℃ to a hentriaconta-circulation, and extended 30 seconds at 70 ℃ the back; The 32 circulation is to extend 7 minutes at 70 ℃;

The scorpion toxin gene that the pcr gene amplified reaction is obtained carries out purifying, and 3.9kb carrier pCR2.1 is gone in this scorpion toxin gene clone obtains the pCR2.1-scorpion toxin, and the exactness of pCR2.1-scorpion toxin gene order is confirmed in order-checking on the nucleotide sequencing instrument;

(2) obtain the scorpion toxin gene fragment with restriction enzyme BglII digestion pCR2.1-scorpion toxin, then the scorpion toxin gene fragment clone is gone into baculovirus transfer vector pAcGP67b and obtain recombinant chou pAcGP67b-scorpion toxin gene;

In silkworm cultured cell, produce recombinant virus by cotransfection with the reorganization of silkworm baculovirus BmNPV dna homology, the method of described cotransfection is: earlier 1 μ g pAcGP67b-scorpion toxin gene DNA and 15 μ gBmNPV DNA are mixed, then add 14 μ l liposome lipofectin, after add sterile purified water again to final volume 40 μ l, at last with the Sf21 culturing cell of this mixed solution cotransfection logarithmic phase after room temperature was cultivated 15 minutes;

With the above-mentioned recombinant virus that produces by cotransfection earlier with the TC-100 culture medium culturing of serum-free 24 hours, after changing the fresh culture that contains 10% foetal calf serum again into and cultivating 5 days based on 27 ℃, collect medium supernatant as virus stock solution used to be used for screening described recombinant virus; Recombinant virus is taken turns plaque select by 3, finally obtains the reorganization BmNPV-scorpion toxin virus solution of high density purifying, and the concentration of this BmNPV-scorpion toxin virus solution is 10 ⁸Pfu/ml;

(3) earlier with 5 age silkworm larva be immersed in the ice-water bath and anaesthetize, adopting hypodermic method then is 10 with concentration ⁶The reorganization BmNPV-scorpion toxin viral suspension of pfu/ml is injected into silkworm larva and infects expression, and every silkworm is injected 20 μ l; Inject after 1 hour and feed mulberry, and raise down at 23-25 ℃ to silkworm larva;

Collector's silkworm larva in 72-96 hour after infection dissect to infect the fatty body of back silkworm, and with the parent material of this fatty body as the purification of Recombinant scorpion toxin; Be in 7.6 the 20mM Tris-HCl damping fluid, to make reorganization scorpion toxin protein binding to chromatography column with the pH that is dissolved in precooling after the described fatty body homogenate through behind the high speed centrifugation supernatant liquor directly being gone up the Ni-NAT affinity column; Be that 7.6 20mMTris-HCl buffer solution elution is removed foreign protein with pH then, go out bonded reorganization scorpion toxin albumen with 250mM imidazole buffer one-step elution at last.

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