WO2012027930A1

WO2012027930A1 - Vector for soluble expression of exogenous protein, preparation and application methods thereof

Info

Publication number: WO2012027930A1
Application number: PCT/CN2010/079239
Authority: WO
Inventors: 张大兵; 梁婉琪; 申惠峰; 袁政
Original assignee: 上海交通大学
Priority date: 2010-08-31
Filing date: 2010-11-29
Publication date: 2012-03-08
Also published as: CN101942478A

Abstract

A vector for soluble expression of exogenous protein, preparation and application methods thereof are provided. The vector is pET30e, the DNA sequence of which is listed as SEQ ID NO:1. The vector is constructed by cloning the encoding sequence of chaperone FaeE into the expression vector pET30e by using restriction endonucleases, in which the encoding sequence of chaperone FaeE is under the control of T7 promoter and lac operator and the 5'end of it is added with E.coli ribosome binding site. Soluble expression of exogenous protein in E.coli can be enhanced by utilizing the vector which is constructed by inserting the gene encoding the exogenous protein into pET30e.

Description

Plasmid for soluble expression of foreign protein and preparation and application method thereof

The invention relates to a plasmid in the field of bioengineering technology and a preparation and application method thereof, in particular to a plasmid for improving the soluble expression of a foreign protein in Escherichia coli by using the enterotoxin-producing Escherichia coli FaeE gene, and preparation and application method thereof .

Background technique

E. coli has the characteristics of thorough understanding, rapid growth, economical culture, high expression level, candidate plasmid and host, and has become the preferred expression system in the field of genetic engineering technology. However, exogenous proteins tend to be easily degraded by host proteases or form inclusion bodies while achieving high levels of expression. At present, there are many studies on the in vitro renaturation of proteins at home and abroad, but the process is often time-consuming, laborious, and uneconomical. Therefore, exploring the soluble expression of foreign proteins in E. coli has high academic value and broad application prospects.

Enhance soluble expression in exogenous E. coli by co-expression of accessory proteins such as the chaperones GroEL/ES and DnaK-DnaJ-GrpE, Trigger factor, Thioredoxin and Foldase Etc. to increase the solubility of foreign proteins. However, molecular chaperones and folding enzymes should be selectively co-expressed according to the characteristics of the foreign protein. For example, co-expression of the heat shock protein GroEIJES in E. coli can increase certain proteins (α-1,6-fucosyltransferase, Bacillus licheniformis cyclodextrin glucanotransferase, glutamate racemase, Solubility of superoxide dismutase, tyrosine kinase, human collagenase, but other proteins (extracellular domain of human type I interferon receptor 2c subunit, phage P22 structural protein, human acid rich cyst A secreted protein) has no solubilizing effect. Another example is that the expression of GroEL/ES and human tyrosine kinase Lck does not increase the solubility of the latter, while thioredoxin can significantly promote the soluble expression of the foreign protein in E. coli. In addition, temperature may also have an important effect on the effect of co-expression of folding accessory proteins. For example, preS2-S'-p-galactosidase is co-expressed with DnaK and DnaJ chaperones, and can increase the expression level of soluble proteins in the range of 30 to 42 ,, while co-expression of GroEL and GroES molecular chaperones can only be in 30 Ό conditions. Get better results. It is worth mentioning that if several folded accessory protein molecules are simultaneously co-expressed, it is possible to obtain a better effect than expressing a certain folded helper protein alone. Jiro et al. co-expressed GroEl/ES, Trx or DsbBD with glutamate racemase, and the yield of active glutamate racemase increased by 2.2 to 2.3 times. In some cases, co-expression of proteins that increase protein solubility and promote the growth of E. coli can also be effective. For example, Kallio et al. co-expressed Vitreoscilla hemoglobin in E. coli and found that the protein significantly promoted the expression of active protein.

FaeE is a molecular chaperone on the enterotoxin-producing E. coli flagin, which forms a heterotrimer with other subunits of flagellin in the form of homodimers in vivo, thereby protecting these subunits from protease degradation. At the same time, these subunits are inhibited from polymerizing in advance and are escorted to transmembrane proteins. Summary of the invention

The present invention provides a plasmid for soluble expression of a foreign protein, and a preparation and application method thereof, aiming at the above-mentioned deficiencies of the prior art, and constructing an E. coli expression vector containing the molecular chaperone FaeE, which can promote the outside Soluble expression of the source protein in E. coli.

The invention is achieved by the following technical solutions:

The present invention relates to a plasmid for the soluble expression of a foreign protein, which is pET30e whose DNA sequence is shown as Seq ID No.

The plasmid comprises a coding sequence of the chaperone FaeE, and comprises a multiple cloning site consisting of BamH I, EcoR I, Sac I, Sal I, Not I, Xho I restriction endonuclease recognition site, and its molecular chaperone FaeE The coding sequence includes the initiation codon ATG and the stop codon TAA, the coding sequence of the chaperone FaeE and the exogenous gene sequence have an E. coli ribosome binding site, the coding sequence of the chaperone FaeE and the exogenous gene sequence. Co-regulated by the T7 promoter and the lac operon, the end of the foreign gene sequence is fused to the six His-tag genes:

The present invention relates to a method for preparing a plasmid for soluble expression of a foreign protein, which comprises ligating an amplification product obtained by PCR amplification of a plasmid template and a primer set with a pMD18-T vector, and transforming Escherichia coli DH5a to obtain Escherichia coli. The clone, the E. coli clone was digested and ligated as a substrate, and the obtained product was transformed into Escherichia coli DH5« to obtain a soluble expression plasmid molecule.

The Escherichia coli DH5a was purchased from Shanghai Yingjun Biotechnology Co., Ltd.:

The pMD18-T carrier is purchased from Dalian Bao Biological Engineering Co., Ltd.;

The PCR amplification of the plasmid template and the primer set refers to:

a) PCR amplification of the F4ac-type ETEC wild-type strain C83907 plasmid with the first primer and the second primer: or b) carrying the plasmid pETctb containing the cholera toxin B subunit (CTB) gene with the third primer and the fourth primer PCR amplification: or

c) The plasmid pET32a-prx-24 containing the peroxidase precursor (PRX) gene was subjected to PCR amplification with the fifth primer and the sixth primer.

The F4ac type ETEC wild strain C83907 was purchased from China Institute of Veterinary Drug Control;

The nucleotide sequence of the first primer is shown in SEQ ID NO. 4, and the sequence comprises an Nde I restriction endonuclease recognition site.

The nucleotide sequence of the second primer is shown in SEQ ID NO. 5, which comprises a BamH I restriction endonuclease recognition site and an E. coli ribosome binding site.

The nucleotide sequence of the third primer is set forth in SEQ ID NO. 6, which comprises a BamH I restriction endonuclease recognition site. The nucleotide sequence of the fourth primer is shown in SEQ ID NO. 7, which comprises an Xho I restriction endonuclease recognition site.

The nucleotide sequence of the fifth primer is shown in SEQ ID NO. 8, which comprises a BamH I restriction endonuclease recognition site.

The nucleotide sequence of the sixth primer is shown in SEQ ID NO. 9, which contains a Xho I restriction endonuclease recognition site.

The enzyme cleavage linkage refers to:

a) Digested with restriction endonucleases Nde I and BamH I to obtain a 740 bp fragment ligated with the pET30a vector digested with restriction endonucleases Nde I and BamH I: or

b) Digested with BamH I and Xho I to obtain a 309 bp fragment ligated with the pET30e vector digested with BamH I and Xho I.

The pET30a plasmid was purchased from Shanghai Meiji Biotechnology Co., Ltd.;

The invention relates to a method for applying the above-mentioned plasmid for soluble expression of a foreign protein, and the obtained soluble expression plasmid molecule is transformed into E. coli BL21 (DE3) competent cells by heat shock method to obtain a recombinant Escherichia coli colony, and the bacteria are obtained by inoculation culture. An increase in soluble expression is achieved after somatic cells.

The E. coli BL21 (DE3) was purchased from Shanghai Yingjun Biotechnology Co., Ltd.:

The E. coli BL21 (DE3) competent cells were prepared by picking a BL21 (DE3) single colony from a freshly activated SOB-M _g solid medium plate in 50 mL of SOB-M _g liquid medium, 37 The culture was vigorously shaken at °C. When OD600 = 0.3, the bacteria were transferred to a sterile, ice-cold 80 mL centrifuge tube, placed on ice for 10 min, and the culture was allowed to cool to zero. C. 5,000 rpm, 4. C, Centrifuge for 5 min, try to pour the culture solution; Add 30 mL of pre-cooled 0.1 M CaC12 solution to suspend the cells: Place on ice for 20 min: 5,000 rpm, 4 ° C, and centrifuge for 5 min. Carefully pour off the supernatant and resuspend the cells in 1/2 original medium volume of 0.1 M CaC12 (20% glycerol added). After the 100μ tube is dispensed directly for conversion or after liquid nitrogen quick freezing, -70. C storage.

The heat shock transformation refers to: taking E. coli BL21 (DE3) competent cells to melt on ice, then adding the plasmid to be transformed, mixing and placing on ice for 30 min _; 42 ° C, heat shock for 90 sec _; heat After stirring, it was rapidly placed on ice to cool lOmin: ΙΟΟμί anti-liquid LB medium was added, and shaken for 37 min at 37 °C _; 200 μL of the bacterial solution was completely coated on solid LB medium supplemented with the corresponding antibiotic: cultured at 37 ° C for 16 hr.

The inoculation culture refers to: picking up recombinant E. coli colonies and adding 3-5 mL of LB medium containing kanamycin 50 mg/L, and incubating at 37 ° C overnight. The overnight culture was added to 100 mL of LB medium containing kanamycin 50 mg/L at a dose of 1:100, and cultured at 37 ° C with shaking to mid-log phase OD595=0.5-0.6, and added at a ratio of 1:1000. 1M IPTG, cultured at 37 ° C for 3-5 h. The cells were collected by centrifugation at 8,000 rpm at 4 ° C for 10 minutes.

The present invention is compared with the prior art The soluble expression of foreign proteins in E. coli can be significantly improved by using the pET30e vector system. In this system, the soluble expression of CTB was increased by 8.85%, the soluble expression of PRX was increased by 13.18%, and the soluble protein expressed was biologically active. At the same time, the vector system can use the multiple cloning sites such as BamH I, EcoR I, Sac I, Sal I, Not I, Xho I to clone the foreign gene into the vector _P ET30e of the present invention, and utilize the 6His-ta at the 3′ end of the vector. _g Realize rapid purification of foreign proteins: The expressed foreign protein is not fused with other large accessory proteins and does not require further processing. Therefore, the system has a promoting effect on the soluble expression of foreign proteins in Escherichia coli, and is of great significance for further analysis of the functions of these proteins.

DRAWINGS

Figure 1 is a schematic representation of the pET30e E. coli expression vector and SDS-PAGE analysis of the expression of the chaperone FaeE in E. coli.

Figure 2 is a schematic view showing the structure of the pET30e vector.

Figure 3 is a schematic diagram showing the structure of the pET30eCTB vector.

Figure 4 is a schematic diagram showing the structure of the pET30ePRX vector.

Figure 5 shows SDS-PAGE and Western blot analysis of pET30CTB and pET30eCTB expression in E. coli. Figure 6 shows SDS-PAGE and Western blot analysis of pET30PRX and pET30ePRX expression in E. coli. Figure 7 shows the GM1 ganglioside ELISA binding assay to analyze the activity of soluble CTB in the pET30e expression system.

Figure 8 shows the peroxidase activity assay and relative activity analysis obtained by the pET30e expression system.

detailed description

The embodiments of the present invention are described in detail below. The present embodiment is implemented on the premise of the technical solution of the present invention, and the detailed implementation manner and the specific operation process are given. However, the protection scope of the present invention is not limited to the following implementation. example.

Example 1

Schematic diagram of pET30e E. coli expression vector and SDS-PAGE analysis of molecular chaperone FaeE expression in E. coli. The chaperone FaeE coding sequence was cloned into the commercial expression vector pET30a by Nde I and Bam HI restriction enzymes. The gene is located under the control of the T7 promoter and the lac operon, and an E. coli ribosome binding site is added to the 5' end of the chaperone coding sequence, after which the cloning of the foreign protein gene can be performed. Cloning sites, including Bam HI, EcoR I, Sac I, Hind III, Not I, Xho I, are 6 histidine coding sequences following the multiple cloning site. The foreign gene is terminated by the T7 terminator. The carrier structure is shown in Figures 1 and 2.

The pET30a and the obtained pET30e E. coli expression vector were transformed into E. coli expression strain BL21 (DE3), and the single colonies of recombinant E. coli pET30a/BL21 (DE3) and pET30e/BL21 (DE3) were contained in 100 ml containing 50 μηηΐ The fresh LB liquid culture solution of kanamycin was shake cultured at 37 ° C, and cultured until the OD590 value was 0.4, and IPTG was added to a final concentration of 1 mmol/1 for induction for 4 hr. Induced and non-induced E. coli cells at 5,000 rpm at 4 °C After centrifugation for 10 min, the collected cell pellet was resuspended in 10 ml/ _g fresh weight (FW) in PBS (pH 7.4) and sonicated on ice. The cell lysate after ultrasound is at 4. Centrifuge at 10,000 rpm for 10 min at C and collect the supernatant. 10 μL of the supernatant was taken for SDS-PAGE electrophoresis, followed by Coomassie staining. The results (Fig. 1B) show that the FaeE expressed in E. coli has a size of 24.8 kDa, which is consistent with its actual size.

Example 2

SDS-PAGE and Western blot analysis of expression of PET30CTB and pET30eCTB in E. coli The coding sequence of the CTB coding sequence was cloned into the same digested pET30e by Bam HI and Xho I to obtain the expression vector pET30eCTB. The structure is shown in Figure 3.

The DNA sequence of the vector ET30eCTB plasmid is shown in Seq ID No. 2, which comprises the coding sequence of the chaperone FaeE and the coding sequence of the cholera toxin B subunit CTB, the cholera toxin B subunit (CTB) coding sequence. It is obtained by classical cholera vibrio (CVC) genomic DNA amplification as a template.

pET30CTB and pET30eCTB were transformed into E. coli expression strain BL21 (DE3) to obtain recombinant E. coli pET30CTB/BL21 (DE3) and pET30eCTB/BL21 (DE3). The method of bacterial culture, protein induction and treatment was the same as in Example 1. The protein was separated by SDS-PAGE, and the protein was transferred to a nitrocellulose membrane (NC) after separation, and then the membrane was multiplied with the first antibody His-ta _g rabbit. The cloned antibody (Proteintech Group, USA) (1:2,000 v/v) was reacted: then reacted with alkaline phosphatase-conjugated anti-rabbit IgG (1:1,000 v/v): after the membrane was rinsed, the The nitroblue tetrazolium (NBT, Gibco) and 5-bromo-2-chloro-3-indolyl phosphate (BCIP, Gibco) substrate coloring reaction solution were subjected to a coloring reaction: after the reaction, the reaction was terminated by using sterilized distilled water. The results of SDS-PAGE (Fig. 5A) showed that both pET30CTB/BL21(DE3) and pET30eCTB/BL21(DE3) could express the 11.9 kDa CTB protein, and the expressed 6His-CTB was mainly expressed in the two recombinant E. coli proteins. Presupposed in the precipitate, that is, mainly in the form of inclusion bodies. The results of Western blot (Fig. 5B) showed that CTB was expressed in the form of inclusion bodies in addition to recombinant E. coli pET30CTB/BL21 (DE3) and pET30eCTB/BL21 (DE3), and also in pET30eCTB/BL21 (DE3). Soluble form of expression.

M-NTA (Nova _g en, USA) purification of His6preSl fusion protein was performed using an M-NTA affinity chromatography column according to the experimental protocol provided by the supplier. The concentration of the collected protein was determined by the Bradford method. The results showed that soluble 6His-CTB expressed by the pET30e E. coli expression vector system accounted for 8.85% of the total soluble protein expressed by recombinant E. coli pET30eCTB/BL21 (DE3). However, the presence of CTB protein was not detected in the soluble protein expressed by recombinant E. coli pET30CTB/BL21 (DE3). That is, the solubility of the expressed CTB can be increased by 8.85% by the pET30e E. coli expression system.

Example 3

SDS-PAGE and Western blot analysis of expression of PET30PRX and pET30EPRX in E. coli The coding sequence of PRX was cloned into the same digested pET30e by Bam HI and Xho I. The expression vector pET30ePRX was obtained, and the vector structure is shown in Fig. 4.

The DNA sequence of the vector ET30ePRX plasmid is shown in Seq ID No. 3, and comprises the coding sequence of the chaperone FaeE and the coding sequence of rice endogenous peroxidase, the rice endogenous peroxidase (PRX). The gene sequence was obtained by amplification of rice genomic DNA as a template.

pET30PRX and pET30EPRX were transformed into E. coli expression strain BL21 (DE3) to obtain recombinant E. coli pET30PRX/BL21 (DE3) and pET30ePRX/BL21 (DE3). The method of bacterial culture, protein induction and treatment was the same as that of Example 1, and the Western blot analysis method was the same as in Example 2. The results of SDS-PAGE (Fig. 6A) showed that both pET30PRX/BL21(DE3) and pET30ePRX/BL21(DE3) can express 37.8 kDa of PRX protein, and the expressed 6His-PRX is mainly expressed in these two recombinant E. coli proteins. Presupposed in the precipitate, that is, mainly in the form of inclusion bodies. The results of Western blot (Fig. 6B) except that PRX was expressed in the form of inclusion bodies in the recombinant E. coli pET30PRX/BL21 (DE3) and pET30ePRX/BL21 (DE3), and also in pET30ePRX/BL21 (DE3) Soluble form of expression. The soluble 6His-PRX was purified and analyzed to obtain 13.18% of the total soluble protein expressed by the soluble protein recombinant E. coli pET30ePRX/BL21(DE3). In the soluble protein expressed by recombinant E. coli pET30PRX/BL21 (DE3), the presence of PRX protein was not detected. That is, the solubility of the expressed PRX can be increased by 13.18% by the pET30e E. coli expression system.

Example 4

The GM1 ganglioside ELISA binding assay was used to analyze the activity of soluble CTB in the pET30e expression system. The biological activity of the CTB protein was analyzed by GM1-ELISA. The following method was used: 96-well microtiter plate was coated with GM1 (Sigma G27641), while galactose (Gal), BSA, and PBS were used as controls. After overnight at 4 ° C, 85 n _{g of} CTB protein was coated with GM1. On the microplate: a His-tag rabbit polyclonal antibody was used as a primary antibody, and alkaline phosphatase-conjugated goat anti-rabbit IgG was used as a secondary antibody to develop color with NPP. After termination of the reaction, a microplate reader was used. ELx800UV, ΒίοΤΕΚ) The light absorption value of each well solution was measured, and the excitation light wavelength was 405 nm.

It can be seen from the results of Fig. 7 that the purified 6His-CTB expressed by the purified pET30e E. coli has strong binding ability to GM1 ganglioside compared with the control 6His protein, and is compatible with the control galactose and BSA. No binding ability. The natural receptor for CTB in the body is ganglioside, and CTB can form a pentameric form to bind to its receptor. The results show that the soluble 6His-CTB can form its natural conformation. So it can be combined with its receptor.

Example 5

Peroxidase activity assay and relative activity analysis obtained by _P ET30e expression system.

The peroxidase activity was analyzed using an Amplex Red peroxidase/peroxidase assay kit (Molecular Probes) by reference to the instructions. A standard curve was prepared using a standard HRP of 0, 25, 37.5, 50, 62.5, 75, 87.5, and 100 mU/mL with an Amplex Red reaction mixture at 30 ° C for 60 min. Use the standard Lines were used to calculate the relative activities of the 6His-PRX and 6His proteins.

From the results of FIG. 8A, pET30e expression system peroxidases catalyze the release of H _₂ 0 ₂ 0 _2, and 6His-PRX activity increase with the extension of the reaction time, although commercial HRP The activity is somewhat different. However, the relative activity of 6His-PRX calculated from the standard curve was 1.00 μυ mL-1 pmol-1, which was significantly different from 6His (P < 0.01) (Fig. 8B).

Example 6

The present invention relates to the above-described method for increasing the expression of a foreign gene in Escherichia coli, and the obtained soluble expression plasmid molecule is transformed into E. coli BL21 (DE3) competent cells by heat shock. Pick up recombinant E. coli colonies. Add 3-5 mL of LB medium containing kanamycin (50 mg/L) and incubate overnight at 37 °C. The overnight culture was added to a lOOmL LB medium containing kanamycin (50 mg/L) at an inoculation amount of 1:100, and cultured at 37 ° C until the middle of the log (OD595 = 0.5 - 0.6). Add 1M IPTG at a ratio of 1:1000 and incubate at 37 °C for 3-5 h. The cells were collected at 4 ° C 8,000 rpm centrifugation lOmin. Soluble analysis and purification of expressed proteins were performed.

The method of preparing competent cells according to: new SOB-M _g on solid medium plates were picked activated BL21 (DE3) single colonies 50mL SOB-M _g in a liquid medium 37. C was shaken vigorously. When OD600 = 0.3, the bacteria were transferred to a sterile, ice-cold 80 mL centrifuge tube and placed on ice for 10 min to cool the culture to 0 °C. 5,000 rpm, 4 ° C, centrifugation for 5 min, try to pour the culture solution; add 30 mL of pre-cooled 0.1 M CaC12 solution to suspend the cells: place on ice for 20 min; 5,000 rpm, 4 ° C, centrifuge for 5 min. Carefully pour off the supernatant and resuspend the cells in 1/2 original medium volume of 0.1 M CaC12 (20% glycerol added). The ΙΟΟμ tube is directly used for transformation or liquid nitrogen after the split, and stored at -70 °C.

The method for transforming the competent cells is: taking competent cells, melting on ice, then adding the plasmid to be transformed, mixing and placing on ice for 30 min: 42 ° C, heat shock for 90 sec: rapid setting after heat shock The ice was cooled on ice for 10 min: ΙΟΟμί anti-liquid LB medium was added, and shaken for 37 min at 37 °C _; 200 μL of the bacterial solution was completely coated on solid LB medium supplemented with the corresponding antibiotic: 37. C culture for 16 hr.

The protein purification method was carried out in accordance with Novagen's The pET System manual. The purified protein was subjected to relevant activity analysis.

The soluble analysis method was as follows: The cells were washed twice with PBS (1 mM KH2P04, 10 mM Na2HP04, 137 mM NaCl, 2.7 mM KC1, pH 7.4), and then the cells were resuspended in PBS and subjected to ultrasonication. It was then centrifuged at 12,000 rpm for 30 min at 4 °C. The supernatant and the pellet were separated, and the pellet was resuspended in an equal volume of PBS and analyzed. The supernatant and the precipitate were separately subjected to SDS-PAGE analysis and Western blot analysis.

The SDS-PAGE analysis method is: adding the collected protein sample to an equal volume of 2xSDS gel loading buffer (50 mM Tis-HCl (pH 6.8), 100 mM dithiothreitol (DTT), 2% ( m/V) SDS (electrophoresis grade), 0.1% bromophenol blue, 10% (v/v) glycerol), boiled for 5-10 min, centrifuged at 12,000 rpm for 5 min, and the supernatant was taken for SDS-PAGE analysis. The Western blot analysis method is as follows: the protein sample is transferred to a nitrocellulose (NC) membrane after SDS-PAGE electrophoresis. 200mA, transfer film for 2hr, turn off the power after transfer, take out the NC membrane and put it into the container (culture dish), add appropriate blocking buffer (PBS buffer containing 5% (w/v) skim milk powder), light at room temperature Incubate for 1-2 hours with gentle shaking. Replace the culture dish with 10 mL of the primary antibody diluted in blocking buffer (1:500 dilution) and incubate at 4 °C for 2 hr. The PBS buffer was washed three times for 10 min each time. The NC membrane was transferred to another dish, and an appropriate amount of secondary antibody buffer (150 mM NaCl, 50 mM Tris-Cl, pH 7.5) was added and washed at room temperature for 10 min. The buffer was decanted, the above secondary antibody buffer containing 5% skim milk powder was added, and a secondary antibody (alkaline phosphatase-conjugated goat anti-rabbit IgG) was added in an amount of 1:5000, and incubated for 1 hr at room temperature with shaking. The NC membrane was transferred to another culture dish, and an appropriate amount of secondary antibody buffer (150 mM NaCl, 50 mM Tris-Cl, pH 7.5) was added thereto, and washed three times at room temperature for 10 minutes each time. Add enzyme-linked antibody chromogenic substrate NBT/BCIP to incubate at room temperature in the dark. After the color of the protein band was reached, transfer the membrane to another dish and stop the reaction with a stop solution (40 μί 0.5Μ ΕΟΤΑ, 10 mL PBS).

The activity analysis method includes the activity analysis of CTB expressed by the system and the activity analysis of PRX. The activity of CTB is analyzed by the method of GM1-ELISA in this embodiment, and the specific method is as follows: 96-well coated with GM1 (Sigma G27641) Enzyme plate (BIO BASIC INC), 2 μ _δ per well (diluted in PBS, 100 pL per well): Simultaneously with galactose (Gal), BSA, PBS as control, 3 replicates per sample, overnight at 4 °C. Discard the coating and rinse the steamed water 4 times continuously: 1% BSA (lg BSA dissolved in 100 mL PBS, pH 7.4) was blocked and allowed to stand at room temperature for 2-5 hr. Discard the blocking solution and rinse the water 4 times: 100n _g CTB protein was coated on the ELISA plate bound to GM1; incubate at 37 °C for 2 hr _; discard the coating and rinse 4 times with distilled water: 300 μί blocking buffer (0.05% Tween 20, 1 mM EDTA, 0.125% BSA, 0.17 M H3B04, 0.12 M NaCl, pH 8.5), 25 was added. C was blocked for 30 min, washed 3 times with distilled water, and then added with 50 μί His-tag antibody diluted in blocking buffer for 2 hr at room temperature: 3 times with distilled water, blocked with 10 min of blocking buffer, and then re-steamed with water. Rinse 3 times, add 50 μί alkaline phosphatase-conjugated secondary antibody diluted in blocking buffer (1: 2000), room temperature 2 hr: 3 times with distilled water, add 75 μί NPP solution [5 mg NPP dissolved in 50 μί The distilled water was diluted with a buffer solution (0.11 M glycine, 1 mM MgC12, 1 M ZnC12, pH 10.4) at a ratio of 1:100. After color development for 1 hour at room temperature, the reaction was terminated by adding 25 μί 0.5 NaOH: The same treatment was carried out for the control. The light absorption value of each well solution was measured with a microplate reader (ELx800UV, BioTEK), and the excitation light wavelength was 405 nm.

The PRX activity was analyzed using an Amplex Red peroxidase fi/peroxidase assay kit (Molecular Probes), and the specific method was referred to the specification. The light absorption value at 560 nm was measured on a SpectraMax 190 (Molecular Devices) instrument using a 1 μ§ purified 6His-PRX protein and Amplex Red reaction mixture at 30 °C. Collected once every lmin, a total of 60min was collected for enzyme activity analysis. 2p _g of 6His protein and 100mU/mL peroxidase (HRP) were used as negative and positive controls, respectively. A standard curve was prepared using a standard HRP of 0, 25, 37.5, 50, 62.5, 75, 87.5, and 100 mU/mL with an Amplex Red reaction mixture at 30 ° C for 60 min. The standard curve was used to calculate the relative activities of the 6His-PRX and 6His proteins.

In summary, by constructing a molecular chaperone subunit FaeE containing the enterotoxin-producing flagellin, Increase the soluble expression of foreign genes in E. coli. The system established by CTB and PRX verification showed that these two proteins could obtain soluble expression in the pET30e vector system, and the soluble CTB expressed could bind to its receptor, which proved that it formed its natural conformation: Soluble PRX also has peroxidase activity.

Therefore, this vector has an important role in enhancing the soluble expression of foreign proteins in Escherichia coli and can be used for the production of various bioengineered enzymes and biomedicine.

Claims

Claim

A plasmid for the soluble expression of a foreign protein, characterized in that the plasmid is pET30e, and the DNA sequence thereof is shown as Seq ID No. 1.

A method for producing a plasmid according to claim 1, wherein the amplification product obtained by PCR amplification of the plasmid template and the primer set is ligated to the pMD18-T cloning vector, and transformed into Escherichia coli DH5a. The E. coli cloning vector was obtained, and the obtained product was transformed into Escherichia coli DH5a by restriction enzyme digestion with E. coli expression vector, and the plasmid pET30e for soluble expression of the foreign protein was obtained.

The method for preparing a plasmid according to claim 2, wherein the PCR amplification of the plasmid template and the primer set is carried out by using any one of the following three methods:

a) PCR amplification of F4ac-type ETEC wild-type strain C83907 plasmid with first primer and second primer: b) PCR of plasmid pETctb containing cholera toxin B subunit (CTB) gene with third primer and fourth primer Amplification:

The method for preparing a plasmid according to claim 3, wherein the nucleotide sequence of the first primer is as shown in SEQ ID NO. 4, and the sequence comprises an Nde I restriction endonuclease recognition. Site.

The method for preparing a plasmid according to claim 3, wherein the nucleotide sequence of the second primer is as shown in SEQ ID NO. 5, and the sequence comprises a BamH I restriction endonuclease recognition. Site and an E. coli ribosome binding site.

The method for preparing a plasmid according to claim 3, wherein the nucleotide sequence of the third primer is as shown in SEQ ID NO. 6, and the sequence comprises a BamH I restriction endonuclease recognition. Site.

The method for producing a plasmid according to claim 3, wherein the fourth primer nucleotide sequence is as shown in SEQ ID NO. 7, and the sequence comprises an Xho I restriction endonuclease recognition site. point.

The method for producing a plasmid according to claim 3, wherein the fifth primer nucleotide sequence is SEQ. As shown in ID NO. 8, this sequence contains a BamH I restriction endonuclease recognition site.

The method for producing a plasmid according to claim 3, wherein the sixth primer nucleotide sequence is as shown in SEQ ID NO. 9, the sequence comprising an Xho I restriction endonuclease recognition site. point.

The method for preparing a plasmid according to claim 2, wherein the enzyme digestion refers to:

A method for applying a plasmid according to any one of the preceding claims, wherein the obtained soluble expression plasmid molecule is transformed into E. coli BL21 (DE3) competent cells by heat shock method to obtain a recombinant Escherichia coli colony. The soluble expression is improved after the cells are cultured by inoculation.

The method for applying a plasmid according to claim 11, wherein the E. coli BL21 (DE3) competent cells are obtained by: picking from a newly activated SOB-M _g solid medium plate. BL21 (DE3) single colony was cultured in 50 mL SOB-M _g liquid medium and cultured vigorously at 37 °C. When OD600=0.3, the bacteria were transferred to a sterile, ice-cold 80 mL centrifuge tube, ice. The lOmin was placed on top to cool the culture to 0 ° C, 5,000 rpm, 4. C, Centrifuge for 5 min, try to pour the culture solution: Add 30 mL of pre-cooled 0.1 M CaC12 solution to suspend the cells: Place on ice for 20 min; 5,000 rpm, 4. C, centrifuge for 5 min, carefully pour off the supernatant, 1/2 original medium volume of 0.1M CaC12 (add 20% glycerol) to resuspend the cells, ΙΟΟμ tube is directly used for transformation or liquid nitrogen after freezing, - Store at 70 ° C.

The method for applying a plasmid according to claim 11, wherein the heat shock transformation refers to: taking E. coli BL21 (DE3) competent cells to be thawed on ice, and then adding the plasmid to be transformed. , mix and place on ice for 30 min;

42 ° C, heat shock 90 sec: heat shock, quickly placed on ice to cool lOmin; add 100pL anti-liquid LB medium, 37V shaking culture for 45min _; 200μί bacteria solution all coated with solid LB medium with corresponding antibiotics Upper; cultured at 37 ° C for 16 hr.

The method for applying a plasmid according to claim 11, wherein the inoculation culture comprises: picking up a recombinant E. coli colony and introducing 3-5 mL of LB medium containing kanamycin 50 mg/L. The culture was shaken overnight at 37 ° C, and the overnight culture was added to 100 mL of LB medium containing kanamycin 50 mg/L at a dose of 1:100, 37. C oscillation culture to logarithm The medium-term OD595 was 0.5-0.6, and 1M IPTG was added at a ratio of 1:1000, and cultured at 37 ° C for 3-5 h, 4. The cells were collected by centrifugation at 8,000 rpm for 10 min.