CN102154359A - New high-efficiency secretion and expression system of colibacillus and application thereof - Google Patents

Abstract

The invention discloses a novel high-efficiency secretion and expression vector jof colibacillus, a preparation method thereof and application thereof. The expression vector is a cyclic plasmid deoxyribonucleic acid (DNA) vector, and comprises a procaryotic copy initial point, a selection marker gene and two series-wound exogenous gene expression box units. Each series-wound exogenous objective (target) gene expression box unit respectively comprises a tac strong promoter, an L-Asparaginasum signal peptide degenerate gene, an exogenous objective (target) gene and a high-transcription terminator rrnBT1T2 which are sequentially arranged with one another. The novel high-efficiency secretion and expression vector disclosed by the invention can secrete and express a polypeptide/protein objective product at a high level in the host cell of the colibacillus, and the expression product can be automatically formed into a correct intramolecular disulfide bond and secreted into cell periplasm/and culture solution, so that the invention is very good for the downstream separation, extraction and purification of the expression product, and has an important application value.

Description

Escherichia coli high-efficiency secretion and expression system and its application

technical field

The invention relates to a high-efficiency secretion expression vector containing two series-connected gene expression cassette units, which is used in the field of genetic engineering and uses Escherichia coli as a host, as well as its preparation method and application.

Background technique

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.

In order to solve this problem, Tan Shuhua et al. (Chinese invention patent 01113526.3) have created a new type of secreted expression vector pTASH, which is characterized in that it contains a strong tac promoter, a degenerate gene of L-asparaginase signal peptide and a high copy of pUC18 The origin of replication, the 3' end of the degenerate gene of the L-asparaginase signal peptide can be combined with the 5' end of the target protein (such as hirudin) coding gene to form a recombinant plasmid, which can secrete and express the gene recombinant hirudin and its mutants.

One of the advantages of the secretory expression vector pTASH is that any foreign gene can be directly spliced with the signal peptide after introducing the NheI site at its 5' end by PCR technology, and there is no redundant connection fragment between the signal peptide gene and the target gene The second advantage is that the high-efficiency secretion and expression of the target protein can be realized, and the expression product has biological activity without denaturation and renaturation.

In order to enhance the secretory expression level, the present invention constructs a more efficient novel Escherichia coli secretory expression vector on the basis of the existing secretory expression vector pTASH (Tan Shuhua et al., Chinese invention patent 01113526.3), which is a circular A plasmid DNA vector containing prokaryotic replication origin, selection marker gene and two tandem exogenous gene expression cassette units. The two tandem exogenous purpose (target) gene expression cassette units each contain a strong tac promoter, an L-asparaginase signal peptide degenerate gene, and an exogenous purpose (target) gene (such as Hirudin gene), strong transcription terminator rrnBT1T2. The new high-efficiency secretion expression vector disclosed by the present invention can secrete and express the target product of polypeptide/protein at a high level in E. coli host cells, and the expression product can automatically form the correct intramolecular disulfide bond, and secrete into the periplasm/and the culture medium outside the cell, It is very beneficial to the downstream separation, extraction and purification of the expression product, and has important application value.

Contents of the invention

The purpose of the present invention is to construct a more efficient novel Escherichia coli secretion expression vector based on the existing secretion expression vector pTASH (Tan Shuhua et al., Chinese invention patent 01113526.3).

The expression vector is a circular plasmid DNA vector, which comprises a prokaryotic replication origin, a screening marker gene and two tandem exogenous gene expression box units. The two tandem exogenous purpose (target) gene expression cassette units each contain a strong tac promoter, an L-asparaginase signal peptide degenerate gene, and an exogenous purpose (target) gene (such as Hirudin gene), strong transcription terminator rrnBT1T2. The transcription directions of the two tandem exogenous gene expression cassette units included can be the same or opposite.

The specific embodiment of the present invention is as follows: take secretory expression carrier pTASH (Tan Shuhua etc., Chinese invention patent 01113526.3) as template, adopt high-fidelity DNA polymerase (as Pfu enzyme), utilize the expression box unit ( Including tac strong promoter, L-asparaginase signal peptide degenerate gene, exogenous target (target) gene, strong transcription terminator rrnBT1T2) amplified, inserted into the BamH I upstream of the expression box unit in the original secretion expression vector pTASH site, thereby forming a more efficient novel E. coli secretion expression vector comprising two tandem exogenous gene expression cassette units, the transcription direction of the two tandem exogenous gene expression cassette units contained therein can be the same or opposite. This plasmid not only has all the advantages of the original plasmid, but also contains two tandem exogenous gene expression cassette units, the transcription intensity of the target gene (target gene) is greatly enhanced compared with the original plasmid pTASH, thereby increasing the expression yield of the target protein .

The novel high-efficiency secretion expression vector according to claim 1 is a circular plasmid DNA vector, which comprises a prokaryotic replication origin, a screening marker gene and two exogenous gene expression cassette units connected in series. The two tandem exogenous purpose (target) gene expression cassette units each contain a strong tac promoter, an L-asparaginase signal peptide degenerate gene, and an exogenous purpose (target) gene (such as Hirudin gene), strong transcription terminator rrnBT1T2. The transcription directions of the two tandem exogenous gene expression cassette units included can be the same or opposite.

In order to amplify the expression cassette unit of the secretion expression vector pTASH (Tan Shuhua et al., Chinese invention patent 01113526.3) (including strong tac promoter, L-asparaginase signal peptide degenerate gene, exogenous target (target) gene, strong transcription termination rrnBT1T2), design and synthesize the following two oligonucleotide PCR primers:

Forward primer pkk223-3TAC-f: 5' GGA TCC AAG CTG TGG TAT GGC TGT GCA GGT CGTAAATC 3' (the underline indicates the BamH I restriction site)

Reverse primer pkk223-3rrnBT2-r: 5' GGA TCC AGC GTT TCT GGG TGA GCAAAAACA GGAAG 3' (the underline indicates the restriction site of BamH I)

Using the original secretory expression vector pTASH (Tan Shuhua et al., Chinese invention patent 01113526.3) as a template, with the above two oligonucleotide chains as primers, under the action of Pfu DNA polymerase, the expression cassette unit (including tac strong promoter, L-asparaginase signal peptide degenerate gene, exogenous target (target) gene, strong transcription terminator rrnBT1T2), the amplified product contains a BamH I restriction site at both ends (GGATCC ). The amplified product was cloned into the pMD-19T SimpleVector vector (product of Treasure Biotech Dalian Co., Ltd., Code No: D104A) using TA cloning technology, and verified by sequencing.

The original secretion expression vector pTASH plasmid was fully digested with BamH I, and the two ends were dephosphorylated with alkaline phosphatase, and the expression cassette unit (including tac strong promoter, L- Asparaginase signal peptide degenerate gene, exogenous target (target) gene, strong transcription terminator rrnBT1T2) were cut out with BamHI, and the fragment was recovered by agarose gel electrophoresis. Then, under the action of T4 DNA ligase, the expression cassette fragment excised by BamH I was ligated with the pTASH plasmid vector fragment that was linearized and dephosphorylated by BamH I, and positive clones were screened out by BamH I enzyme digestion screening technology. Thus, a novel high-efficiency secreted expression vector comprising a prokaryotic replication initiation origin, a selection marker gene and two exogenous gene expression cassette units connected in series is obtained. The two tandem exogenous purpose (target) gene expression cassette units each contain a strong tac promoter, an L-asparaginase signal peptide degenerate gene, and an exogenous purpose (target) gene (such as Hirudin gene), strong transcription terminator rrnBT1T2. The transcription directions of the two tandem exogenous gene expression cassette units included can be the same or opposite.

Another object of the present invention is to disclose an application method of a recombinant plasmid for the production of recombinant hirudin or its mutants, which is characterized in that: the recombinant hirudin or its mutants are secreted and expressed by using the recombinant hirudin or its mutants according to claim 1.

Description of drawings:

Figure 1 pTASH plasmid map

Figure 2 pMD-19TpTASH plasmid map

Figure 3p(TASH) ₂ plasmid map (A is forward insertion, B is reverse insertion)

Figure 4 The activity data of recombinant hirudin III double expression cassette clone p(TASH) ₂ secreted and expressed recombinant hirudin III

Figure 5 Comparison of secreted expression levels of recombinant hirudin III double expression cassette clone p(TASH) ₂ and single expression cassette clone pTASH

Detailed ways

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).

Amplification of expression cassette gene in embodiment 1 expression vector pTASH

Extract the template plasmid pTASH from the original Escherichia coli strain containing the pTASH plasmid (Tan Shuhua et al., Chinese invention patent 01113526.3), and design and synthesize the following primers:

Forward primer pkk223-3TAC-f: 5' GGA TCC AAG CTG TGG TAT GGC TGT GCA GGT CGTAAATC 3' (the underline indicates the BamH I restriction site)

Reverse primer pkk223-3rrnBT2-r: 5' GGA TCC AGC GTT TCT GGG TGA GCA AAA ACA GGAAG 3' (underline indicates BamH I restriction site)

Using the plasmid pTASH as a template, the polymerase chain reaction was carried out under the action of Pfu DNA polymerase. The composition of the 50 μl reaction system is as follows: each 20 pmol of forward primer and reverse primer; 5 units of Pfu DNA polymerase; 5 μl of 10× reaction buffer; 1 μl of dNTP (10 mM each); 3 μl of MgCl ₂ (25 mM); 1 μl of plasmid DNA; Make up to 50 μl with sterile water. The polymerase chain reaction is as follows: denaturation at 95°C for 5 minutes; entering cycle reaction: denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 2 minutes, and 30 cycles of reaction; extension at 72°C for 10 minutes.

The polymerase chain reaction product was separated by 1.5% agarose gel electrophoresis, and DL2,000 ^TM DNA Marker was used as a control to detect the correct size of the gene fragment, and then recover the target fragment. The target fragment is connected with the T vector under the action of T4DNA ligase, and the connection material is transformed into Escherichia coli host bacteria. A plasmid was extracted from the positive clone, and the sequence was confirmed to be correct by sequencing. This plasmid was named pMD-19T-TASH.

Example 2 Construction of Double Expression Cassette Recombinant Hirudin Efficient Secretion Expression Plasmid

The plasmid pMD-19T-TASH was extracted by alkaline lysis, digested with BamH I, and DL2,000 ^TM DNA Marker was used as a control, separated by 1.5% agarose gel electrophoresis, and the digested fragment was recovered to obtain an expression of about 1.1kb box fragment.

Plasmid pTASH was extracted from the original strain and digested with BamHI to obtain linearized pTASH with the same cohesive ends as the expression cassette fragment. To prevent linearized pTASH from self-cyclization during the ligation reaction, its 5' phosphate group needs to be converted to a hydroxyl group using the dephosphatase CIAP. The 40 μl reaction system is as follows: 4 μl of 10× reaction buffer, 2 μl of CIAP, 34 μl of linearized pTASH, reacted at 37°C for 4 hours; inactivated at 65°C for 30 minutes to terminate the reaction, and then precipitated with ethanol to obtain relatively pure dephosphorylated linearized pTASH. pTASH.

The expression cassette fragment and the dephosphorylated linear pTASH were ligated overnight at 16° C. under the action of T4 DNA ligase, and the ligated product was directly transformed into E. coli host cells.

The double expression cassette plasmid was screened using the BamH I enzyme digestion method. Since both ends of the newly inserted expression cassette are BamH I restriction sites, the clone with a 1.1 kb size fragment after digestion is a double expression cassette positive clone.

In addition, in order to detect the insertion direction of the newly inserted expression cassette, EcoR I enzyme digestion can be used for verification. If it is inserted in the forward direction, a fragment with a size of 1162bp can be cut out; if it is inserted in the reverse direction, a fragment with a size of 462bp can be cut out. fragments. The digested products were electrophoresed on 1% agarose gel, and DL2,000 ^TM DNAMarker was used as a control.

Example 3 High-efficiency secretory expression of recombinant hirudin III

The selected expression cassette was inserted into the recombinant hirudin III double expression cassette clone p(TASH) ₂ inserted in the forward direction, inserted into 30ml LBA (tryptone 1%, yeast powder 0.5%, NaCl 1%, Amp100μg/ml, pH7.0 ) liquid culture medium to carry out seed liquid culture, the culture condition is 37 ℃ 220rpm vibration culture 14 hours, transfer to fermentation medium (tryptone 1%, yeast powder 0.5%, sodium glutamate 4% with 2% inoculum size then) , malt powder 1.0%, Amp100μg/ml, KH ₂ PO ₄ 0.374%, Na ₂ HPO ₄ 12H ₂ O 1.75%, pH 7.0), cultured at 37°C and 220rpm for 24h, and measured anticoagulant blood in the supernatant of the fermentation broth Enzyme activity.

Hirudin bioactivity assay was carried out with reference to Markwardt's thrombin titration method (F.Markwardt, Hirudin as an inhibitor of thrombin, Methods Enzymol.19 (1970) 924-932.): Add 200 μl of 0.5% cow's milk in the small hole of the microtiter plate Blood fibrinogen (pH7.4, prepared in 50mM Tris-HCl buffer), then add 10-100μl hirudin solution, and mix well. Draw the standard thrombin solution (100 NIH unit/ml) with a micro-sampler for titration, each titration is 5 μl (0.5 NIH unit), and the time interval is 1 min. If the fibrinogen coagulates within 1 min, it means that the fibrinogen has been coagulated. end. Since hirudin and thrombin are combined 1:1, each consumed thrombin unit (NIHU) is equivalent to one antithrombin unit (ATU). Therefore, the number of units of hirudin can be converted from the consumption of thrombin.

Double expression cassette recombinant hirudin IIIp (TASH) ₂ clone secretion expression activity assay results (see Table 1):

Table 1-1. The first set of data of double expression cassette activity assay

Table 1-2. The second set of data of double expression cassette activity assay

Table 1-3. The third set of data of double expression cassette activity assay

The results of the secretory expression activity assay of the pTASH recombinant clone of the single expression cassette strain (see Table 2):

Table 2-1. The first set of data of single expression cassette activity assay

Table 2-2. The second set of data of single expression cassette activity assay

Table 2-3. The third group of data of single expression cassette activity assay

sequence listing

SEQUENCE LISTING

<110> China Pharmaceutical University

<120> Escherichia coli high-efficiency secretion and expression system and its application

<140>201010617881.3

<141>2010-12-31

<160>2

<170>PatentIn version 3.5

<210>1

<211>38

<212>DNA

<213> Artificial sequence

<220>

<223>The forward primer designed and synthesized by using the plasmid pTASH as a template to amplify the target gene fragment is named

pkk223-3TAC-f.

<400>1

ggatccaagc tgtggtatgg ctgtgcaggt cgtaaatc 38

<210>2

<211>35

<212>DNA

<213> Artificial sequence

<220>

<223>The reverse primer designed and synthesized by using the plasmid pTASH as a template to amplify the target gene fragment is named

pkk223-3rrnBT2-r.

<400>2

ggatccagcg tttctgggtg agcaaaaaca ggaag 35

Claims (4)

1. a cyclic plasmid dna vector comprises protokaryon ori, selection markers gene and two placed in-line exogenous gene expression housing units.Described two placed in-line external source purposes (target) genetic expression housing unit contains tac strong promoter, L-Asparaginase signal peptide degeneracy gene, external source purpose (target) gene, the strong transcription terminator rrnBT1T2 that arranges successively respectively.

2. carrier according to claim 1, it is characterized in that two placed in-line exogenous gene expression housing unit transcriptional orientations being comprised can be identical also can be opposite.

3. carrier according to claim 1 and 2 is characterized in that containing the high copy of pUC18 replication origin.

4. application that is used for the intestinal bacteria efficient secretory expression carrier of recombinant protein or polypeptide production at gene recombination r-hirudin and mutant thereof, it is characterized in that: application rights requires plasmid vector secreting, expressing gene recombination r-hirudin and the mutant thereof described in 1.

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