CN1319671A - Novel secretion expression vector and its application of recombinant hirudin expression technology - Google Patents

Abstract

A high-efficiency secretion expression vector based on a new degenerate gene of E. coli (E.coli) L-asparaginase signal peptide and its application in expressing recombinant hirudin. The vector includes a strong tac promoter, L-aspartic acid A novel degenerate gene for amidase signal peptide and a high-copy origin of replication in pUC18. The target gene is placed downstream of the signal peptide gene for expression, and the target product can form the correct spatial conformation and be secreted into the periplasm or/and culture medium of the cell at the same time. The present invention ensures intramolecular disulfide bonds when expressing bioactive proteins or polypeptides. It is of great significance to facilitate the downstream extraction and purification of the expression product.

Description

New Secretion Expression Vector and Its Application in Recombinant Hirudin Expression Technology

The invention relates to the establishment and practical application of a novel Escherichia coli high-efficiency secretion expression vector applied in the field of genetic engineering, especially the application in the expression technology of recombinant hirudin.

There is no general rule for the expression of polypeptide proteins in genetic engineering technology. In order to obtain effective expression, different expression systems and expression methods are adopted for different target products due to their different structural properties. Escherichia coli is the earliest prokaryotic expression system, and its expression methods mainly have three forms: non-fusion expression, fusion expression and secretory expression. Misfolding of peptide chains and mismatching of disulfide bonds during the renaturation process of non-fusion expression methods often lead to a greatly reduced yield of active products. In addition, due to the inevitable addition of a methionine at its N-terminus, this will seriously affect the biological activity of some polypeptide molecules. Although the fusion protein is easy to be expressed at a high level, there are certain problems in the purification and cleavage of the fusion protein. It is difficult to restore its natural conformation by chemical cleavage; while the cleavage efficiency of enzymatic cleavage is not high, and the final yield of the product is low. The secretory expression method is to chimerize the target gene downstream of the signal peptide gene, and the target product is secreted into the periplasm or culture medium mediated by the signal peptide. active peptide products.

Existing secretory expression vectors, such as PIN-Ⅲ-ompA, adopt the Escherichia coli outer membrane protein (ompA) signal peptide sequence, followed by a multiple cloning site for inserting foreign genes. Finally, there is often a redundant linking sequence between the signal peptide gene and the target gene. In this way, in order to obtain the correct target product at the N-terminal, site-directed mutagenesis must be used to remove the redundant sequence between the signal peptide gene and the target gene. Very complicated.

The purpose of the present invention is to create a novel secretory expression vector. One of the advantages of this vector is that any foreign gene can be directly connected with the signal peptide after introducing the NheI site at its 5' end by PCR technology, and the signal peptide gene There is no redundant connecting segment with the target gene; the second advantage is that it can achieve high-efficiency secretion and expression of the target protein, and the expressed product has biological activity without denaturation and renaturation.

The object of the present invention is also to set up a new type of secretion expression vector capable of secreting the expression product into the periplasm or medium while maintaining its biological activity, and its application in realizing the secretion expression of the target gene, especially in the gene Application of recombinant hirudin and its mutants.

Embodiments of the present invention are as follows:

A prokaryotic secretory expression vector for recombinant protein or polypeptide production is characterized in that it contains a strong tac promoter, a degenerate gene of L-asparaginase signal peptide and a high-copy replication origin of pUC18.

An application of a prokaryotic secretory expression vector for recombinant protein or polypeptide production, characterized in that: a prokaryotic secretory expression vector containing a strong tac promoter, an L-asparaginase signal peptide degenerate gene and a pUC18 high-copy replication origin The 3' end of the L-asparaginase signal peptide degenerate gene and the 5' end of the target protein coding gene are spliced together to form a recombinant plasmid. After the plasmid is introduced into the E.coli host cell, the secreted expression of the target gene can be realized.

The application of a prokaryotic secretory expression vector for recombinant protein or polypeptide production in gene recombinant hirudin and its mutants is characterized in that: a degenerate gene containing a strong tac promoter, an L-asparaginase signal peptide and The 3' end of the degenerate gene of the L-asparaginase signal peptide in the prokaryotic secretion expression vector of the pUC18 high-copy replication origin and the 5' end of the target protein hirudin coding gene are spliced together to form a recombinant plasmid, which secretes and expresses the gene recombinant leech Elements and their mutants.

The basic strategy of this type of vector design is to use codons according to the L-asparaginase signal peptide amino acid sequence and gene sequence (Jennings M P and Beacham I R.1990.Journal of Bacteriology.172(3):1491-1498.) The purpose of designing a new signal peptide degenerate gene is to introduce the NheI restriction enzyme cutting site into the 3' end gene of the signal peptide coding gene, so as to use this site to splice with the target protein coding gene while ensuring The reading frame remains unchanged; the signal peptide-target protein fusion gene is inserted into the multiple cloning site downstream of the expression vector promoter to construct a recombinant secretory expression vector expressing the target protein.

Design and Synthesis of Signal Peptide Degenerate Genes

The original AsPsⅡ signal peptide gene sequence (Jennings M P and Beacham I R.1990. Journal of Bacteriology.172(3):1491-1498.) is: Met Glu Phe Phe Lys Lys Thr Ala Leu Ala Ala Ala Leu Val Met GlyATG GAG TTT TTC AAA AAG ACG GCA CTT GCC GCA CTG GTT ATG GGTTAC CTC AAA AAG TTT TTC TGC CGT GAA CGG CGT GAC CAA TAC CCAPhe Ser Gly Ala Ala Leu AlaTTT ACT GGT GCA GCA TTG GCAAAA CGTAAA TCA CCA A CGT

For cloning, it is necessary to use the degeneracy of codons to design it as the following degenerate gene: Met Glu Phe Phe Lys Lys Thr Ala Leu Ala Ala Leu Val Met GlyATG GAG TTT TTC AAA AAG ACG GCA CTT GCC GCA CTG GTT ATG GGTTAC CTC AAA AAG TTT TTC TGC CGT GAA CGG CGT GAC CAA TAC CCAPhe Ser Gly Ala Ala Leu AlaTTT AGT GGT GCA GCG CTA GCTAAA TCA CCA CGT CGC GATCGA

In this way, the NheI restriction endonuclease site GCTAGC was introduced at the 3' end of the coding gene without changing the original amino acid sequence of the AsPsII signal peptide.

In order to assemble the above-mentioned AsPsII signal peptide degenerate gene, the following four oligonucleotides were synthesized:

S1-(1): 5’ AA TTC ATG GAG TTT TTC AAA AAG ACG GCA CTT GC 3’

S1-(2): 5’ TGC GGC AAG TGC CGT CTT TTT GAA AAA CTC CAT G 3’

S2-(1): 5’ C GCA CTG GTT ATG GGT TTT AGT GGT GCA GCG 3’

S2-(2): 5’ C TAG CGC TGC ACC ACT AAA ACC CAT AAC CAG 3’

S1-(1) and S1-(2) as one group; S2-(1) and S2-(2) as another group were annealed and paired respectively, phosphorylated by T4 polynucleotide kinase, and then carried out by T4 DNA ligase Link to get AsPsⅡ signal peptide degenerate gene with EcoRI at the 5' end and NheI restriction endonuclease sticky end at the 3' end: 5'AAT TTC ATG GAG TTT TTC AAA AAG ACG GCA CTT GCC GCA CTG GTT

3’G TAC CTC AAA AAG TTT TTC TGC CGT GAA CGG CGT GAC CAAATG GGT TTT AGT GGT GCA GCG 3’TAC CCA AAA TCA CCA CGT CGC GAT C 5’

The NheI site can be introduced into the 5' end of the HV3 gene by using PCR technology, and after digestion, it is connected with the above-mentioned signal peptide gene to obtain the L-asparaginase signal peptide-hirudin fusion gene.

Use NruI and PvuI to cut out the fragment containing the promoter and multiple cloning site of plasmid pkk223-3; use PvuII and PvuI to cut out the fragment containing the origin of replication of plasmid pvc18, and connect the above two fragments to obtain plasmid pTA. The hirudin secretion expression plasmid pTASH is obtained by inserting the L-asparaginase signal peptide-hirudin fusion gene into the multiple cloning site of the plasmid pTA.

The beneficial effects of the present invention are as follows: the secretory expression vector in the present invention has two major advantages. One is that any foreign gene can be directly spliced with the signal peptide after being introduced into the NheI site at its 5' end by PCR technology. There is no redundant connecting segment between the signal peptide gene and the target gene; second, efficient secretion and expression are achieved, and the expression product has biological activity without denaturation and renaturation.

In the present invention, the target gene is conveniently placed downstream of the signal peptide gene and expressed, and the expression level of the target product is relatively high; the expression product avoids the extension of N-terminal methionine; Sulfur bond mismatch problem; the expression product directly forms an active molecule with a natural conformation during the secretion process, and is secreted into the periplasm or culture medium, eliminating the need for complex product post-processing procedures such as denaturation, cutting, and renaturation of the product , which reduces the loss of the product and brings great convenience to the separation and purification of the target product.

Graphic description:

Fig. 1 Hirudin HV3 coding gene sequence in recombinant plasmid pUCH33

Figure 2 pTASH plasmid map (Ptac: tac promoter; SIG: L-asparaginase signal peptide gene; HV: hirudin III coding gene; AP: ampicillin resistance gene; Ori: pUC18 high copy origin of replication)

The following preferred examples describe the present invention in detail, but are not meant to limit the scope of the present invention. The conventional molecular cloning operations in the examples refer to the literature ("Molecular Cloning Experiment Guide" Second Edition, translated by Jin Dongyan et al., 1995, Science Press).

Example 1 Assembly of L-asparaginase degenerate signal peptide gene

The 5' end of the oligonucleotide chain synthesized by the DNA synthesizer is a hydroxyl group, which is phosphorylated with T4 polynucleotide kinase before annealing and ligation. The phosphorylation reaction was carried out according to the product manual of Promega Company, and the reaction time was appropriately prolonged in order to reduce the amount of kinase used. The composition of the 20ul reaction system is as follows: oligonucleotide chain 200pmol; 10×reaction buffer 2ul; ATP (5mM) 1ul; appropriate amount of H ₂ O; T4 polynucleotide kinase 2ul. React at 37°C for 2.0 hours, then incubate at 70°C for 10 minutes to stop the enzyme reaction.

Mix equimolar complementary oligonucleotide chains in 80 ul final volume of annealing buffer and cover with sterile paraffin oil, keep warm in a 95°C water bath for 5 minutes, and then slowly cool down to room temperature. After precipitating the annealed mixture with ethanol, use vertical plate non-denaturing polyacrylamide gel electrophoresis, the gel concentration is 5-15%, the electrophoresis buffer is 1×TBE, and the voltage is 8v/cm, and pGEM-3Zf(+ )/HaeⅢ DNA Marker as the standard, recovered after electrophoresis, and ligated with T4 DNA ligase to obtain a degenerate signal peptide gene with a size of about 66bp. Example 2 Preparation of L-asparaginase signal peptide-hirudin fusion gene

Design the following pair of primers:

(1) 5'AATGCTAGCTATCACCTACACTGACTGCACC 3' (Primer 1)

(2) 5'CCCAGTCACGACGTTGTAAAACG 3' (Primer 2, GIBCOBRL company pUC universal sequencing primer sequence)

Plasmid pUCH33 (a recombinant plasmid containing hirudin HV3 coding gene cloned by artificial synthesis technology in our laboratory, the gene is located between the SacⅠ/HindIII restriction sites of pUC18, the sequence is shown in Figure 1) was used as a template for PCR amplification, and the amplified The amplified fragment was double-digested with NheI and HindIII to obtain the hirudin HV3 coding gene with NheI sticky end at the 5' end and HindIII sticky end at the 3' end. T4 DNA ligase was used to connect the gene with the L-asparaginase degenerate signal peptide gene obtained in Example 1 to obtain the L-asparaginase signal peptide-hirudin fusion gene.

Example 3 Construction of Secreted Expression Vector

Take the plasmid pkk223-3 (see Brosius, J.Proc.Acad.Sci.USA 1984,81:6929) and digest it with NruI and PvuI. The digested product is separated by agarose gel electrophoresis, and the fragment containing the tac promoter is recovered (Fragment 1); Plasmid pUC18 (purchased from Shanghai Huamei Bioengineering Co., Ltd.) was double digested with PvuII and PvuI, and the digested products were separated by agarose gel electrophoresis, and the fragment (fragment 2) containing the origin of replication of pUC8 was recovered. Ligate fragment 1 and fragment 2 with T4 DNA ligase, transform the ligation product into E.coliDH5α to obtain plasmid pTA, insert the L-asparaginase signal peptide-hirudin fusion gene prepared in Example 2 into plasmid pTA to obtain recombinant plasmid pTASH.

Secretion and expression of embodiment 4 recombinant hirudin HV3

Transform the recombinant plasmid pTASH into E.coli AS1.357, pick positive clones, insert into 50ml LBA liquid medium (1% peptone, 0.5% yeast extract, 1% sodium chloride, 100ppm ampicillin, pH7.0) at 37°C Shaking culture was obtained for 16 hours to obtain a seed liquid, and this liquid was inserted into a fermentation medium (corn steep liquor 5.0%, beef extract 3.5%, sodium glutamate 1.0% by 2% (V/V) ratio. Ampicillin 100ppm, pH7. 0) shake the flask at 37°C for 20 hours, and the antithrombin activity in the supernatant of the fermentation broth reaches 700 ATU/ml of fermentation broth.

The bioactivity of hirudin was determined with reference to Markwardt's thrombin titration method (Markwardt FMethods in Enzymology 1970, 19:924-932). Add 200ul 0.5% bovine fibrinogen (prepared in 50mM Tris-HCl buffer (pH7.4)) to the small hole of the microtiter plate, then add 10-100ul hirudin solution, and mix well. Draw a standard thrombin solution (100 NIH unit/ml) with a micro-sampler for titration, each titration is 5ul (0.5 NIH unit), and the time interval is 1 minute. If the fibrinogen coagulates within 1 minute, that is Indicates that the titration end point has been reached. The number of units of hirudin can be converted from the consumption of thrombin. Since hirudin and thrombin are combined 1:1, each consumed thrombin unit (NIHU) is equivalent to one antithrombin unit (ATU).

Claims (3)

1, a kind of prokaryotic secretion expression carrier that is used for recombinant protein or polypeptide production is characterized in that: contain the high copy of tac strong promoter, L-Asparaginase signal peptide degeneracy gene and pUC18 replication origin.

2, a kind of application that is used for the prokaryotic secretion expression carrier of recombinant protein or polypeptide production, it is characterized in that: connect with target protein encoding gene 5 ' end matching with L-Asparaginase signal peptide degeneracy gene 3 ' end in the prokaryotic secretion expression carrier that contains the high copy of tac strong promoter, L-Asparaginase signal peptide degeneracy gene and pUC18 replication origin and set up into recombinant plasmid, after this plasmid imported the E.coli host cell, goal gene carried out secreting, expressing.

3, a kind of application that is used for the prokaryotic secretion expression carrier of recombinant protein or polypeptide production at gene recombination r-hirudin and mutant thereof, it is characterized in that: application rights requires 2 described plasmid vector secreting, expressing gene recombination r-hirudin and mutant thereof.

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