High acid-base tolerant fusion protein and preparation method and application thereof

Abstract

The application provides a fusion protein PA-G which fuses Ig dominant binding domains of protein A and protein G, and the amino acid sequence of the fusion protein PA-G is as shown in SEQ ID NO. 1. The fusion protein PA-G disclosed by the application has the immunoglobulin binding capacity, has the larger biological characteristic potential than protein A and protein G, has the better affinity and faster binding efficiency for different species and subtypes of immunoglobulins, and through acid and alkali resistance research experiments, it is found that the most important thing of the fusion protein PA-G is that the pH tolerance is wider, and the application prospect and application range are wider.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering, in particular to a fusion protein with high acid-base tolerance and a preparation method and application thereof. BACKGROUND

[0002] Several proteins with inherent affinity for immunoglobulin (Ig) have been isolated from various bacteria. These molecules include protein A extracted from Staphylococcus aureus, and protein G from group C streptococci; they all contain 55-76 repeating amino acid residues that can mediate Ig binding. Protein A is an immunoglobulin-binding protein of the cell wall of Staphylococcus aureus, which contains five Ig-binding domains. Protein A can specifically bind to the Fc region of IgG and also to the Fab region of IgG. Protein G is an immunoglobulin-binding protein of the cell wall of streptococci, which contains three domains in tandem with IgG-specific structure. Similarly, protein G also has the property of partially binding to the CH1 domain of Fab.

[0003] Protein A and protein G are natural proteins of microbial origin that can bind to mammalian immunoglobulins. Protein G not only binds to the Fc region, but also binds to the CH1 domain of human IgG1 isotype. Therefore, it has a wider application compared to protein A. The ability of protein A and protein G to maintain their functions when coupled with fluorescent pigments, enzymes or gold particles makes them extremely valuable detection reagents in ELISA, immunohistochemistry, flow cytometry and electron microscopy.

[0004] Based on these properties, protein A and protein G have great advantages in the fields of antibody purification, immunoprecipitation, effective removal or reduction of non-specific background, etc. The binding capacity of protein A and protein G to different immunoglobulins is not the same, and it is affected by the source and subclass of the latter. The separate protein A or protein G on the market cannot meet the binding of immunoglobulins of most species, resulting in a single type of purified antibody. If separate protein A or protein G is used instead of secondary antibody and widely used in the field of immunochemistry, it may cause a decrease in the stability of the detection results. If it is applied in the field of molecular research, it may not completely remove the background of some experimental results, causing misjudgment of the results.

[0005] The patent document with publication number CN102676562A and the title of "Preparation method and application of a fusion protein with broad spectrum adsorption capacity for antibodies" discloses a fusion protein obtained by genetically engineering the genes of protein A and protein G, which has the advantages of protein A and protein G, and can selectively bind to different subclasses of human immunoglobulin G and has low non-specific adsorption of non-immunoglobulin substances such as albumin in blood. SUMMARY

[0006] In view of the above technical limitations, the present application provides a high acid-base tolerant fusion protein, a preparation method and application thereof.

[0007] To achieve the above object, the present application adopts the following technical solution:

[0008] The application point of the present application is to provide a fusion protein PA-G, which fuses the Ig dominant binding domain of protein A and protein G, and the amino acid sequence of the fusion protein PA-G is the sequence shown in SEQ ID NO. 1, or a sequence having more than 90% homology with the sequence shown in SEQ ID NO. 1 and having the same function.

[0009] The amino acid sequence shown in SEQ ID NO. 1 is:

[0010] AQHDEAQQNAFYQVLNMPNLNADQRNGFIQSLKDDPSQSANVLGE AQKLNDSQAPKADAQQNNFNKDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVLGEAKKLNESQAPKADNNFNKEQQNAFYEILNMPNLNEEQRNGFIQSLKDDPSQSANLLSEAKKLNESQAPKADNKFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFNKEQQNAFYEILHLPNLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNDAGGGGSGGGGSGGGGSGGGGSDTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEKPEVIDASELTPAVTTYKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEKPEVIDASELTPAVTTYKLVINGKTLKGETTTKAVDAETAEKAFKQYANDNGVDGVWTYDDATKTFTVTEMVC*.

[0011] The second application point of the present application is to provide a gene encoding the above-mentioned fusion protein PA-G, and the nucleotide sequence of the encoding gene is the sequence shown in SEQ ID NO. 2, or a sequence having more than 90% homology with the sequence shown in SEQ ID NO. 2.

[0012] The nucleotide sequence represented by SEQ ID NO. 2 is:

[0013]

[0014] The third aspect of the present application provides a recombinant expression vector into which the gene is inserted.

[0015] Optionally, the recombinant expression vector is a pET series vector, preferably a pET-32a expression plasmid, a pET-26b expression plasmid or a pET-28a expression plasmid, and most preferably a pET-28a expression plasmid.

[0016] The fourth aspect of the present application provides a recombinant strain of the recombinant expression fusion protein PA-G, which is obtained by transforming the Escherichia coli competent cell with the recombinant expression vector.

[0017] Optionally, the recombinant strain of the recombinant expression fusion protein PA-G is an Escherichia coli BL21 competent cell, an Escherichia coli rosetta competent cell or an Escherichia coli DH5a competent cell, and preferably an Escherichia coli BL21 competent cell.

[0018] The fifth aspect of the present application provides a method for preparing the fusion protein PA-G, which is prepared by fermentation of the recombinant strain.

[0019] Optionally, the method comprises the following steps: constructing a recombinant expression vector containing the gene of the fusion protein PA-G by using the expression vector pET-28a; transforming the Escherichia coli DH5a competent cell with the vector, and obtaining the recombinant strain capable of expressing the fusion protein PA-G by culturing and screening; inducing protein expression by IPTG after culturing the recombinant strain, and separating and purifying the expressed fusion protein PA-G.

[0020] The sixth aspect of the present application provides the application of the fusion protein PA-G in antibody purification, immunoprecipitation and expanding acid-base tolerance.

[0021] In the application of antibody purification, based on the popular monoclonal antibody (mAbs), the largest biopharmaceutical category in clinical trials is rapidly increasing, and effective removal of host cell proteins, cell culture medium additives, DNA and endotoxins from monoclonal antibody preparations can make the protein safely applied to human therapy.

[0022] In the application of immunoprecipitation, the specific reaction of the antibody can be used to purify and enrich the target protein for identification and identification of a certain type of protein.

[0023] In the application of expanding acid-base tolerance, the threshold of the characteristics of adapting to polar substances is increased, and the range of pH of the used buffer is expanded.

[0024] The seventh application of the application provides the coding gene of the fusion protein PA-G, the recombinant expression vector, and the recombinant strain in the application of antibody purification, immunoprecipitation, and expanding acid-base tolerance.

[0025] Compared with the prior art, the application has the following advantages:

[0026] The application discloses a fusion protein PA-G with immunoglobulin binding capacity, which is constructed to overcome the fact that the existing protein A and protein G cannot cover all different subtype and source immunoglobulins, and has better physical and chemical properties. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 It is a protein expression condition optimization diagram of the fusion protein PA-G (Protein AG) of the application, in which lane M is 100kDa color pre-dye protein; lanes 1-4 are all 2h of induction time, and the induction agent concentrations are 1, 0.8, 0.4 and 0.2mmol / L respectively; lanes 5-8 are all 3h of induction time, and the induction agent concentrations are 1, 0.8, 0.4 and 0.2mmol / L respectively; lanes 9-12 are all 4h of induction time, and the induction agent concentrations are 1, 0.8, 0.4 and 0.2mmol / L respectively; and lane 13 is 1h of induction time and 1mmol / L of induction agent concentration.

[0028] Figure 2 It is a protein expression form analysis diagram of the fusion protein PA-G (Protein AG) of the application, in which lane M is 100kDa protein Maker.

[0029] Figure 3The figure of protein purification condition optimization for the fusion protein PA-G (Protein AG) of the application; wherein, lane M: 120 kDa protein Maker.

[0030] Figure 4 The figure of ELISA data for the fusion protein PA-G (Protein AG) of the application combined with goat anti-mouse (IgG).

[0031] Figure 5 The figure of SDS-PAGE detection result for the acid and alkali resistance of the fusion protein PA-G (Protein AG) of the application.

[0032] Figure 6 The figure of ELISA data result for the acid and alkali resistance of the fusion protein PA-G (Protein AG) of the application.

[0033] Figure 7 The figure of plasmid structure of the expression vector constructed by the application.

[0034] Figure 8 The figure of the secondary and tertiary structure of the protein PA-G of the application.

[0035] Figure 9 The figure of gene sequence optimization of the fusion protein of the application, showing the before-and-after comparison of codon adaptation index (CAI), relative synonymous codon usage (RSCU) and GC content. DETAILED DESCRIPTION

[0036] In order to make the objects, technical solutions and advantages of the application clearer and more comprehensible, the application will be further described in detail below. However, it should be understood that the description herein is only used to explain the application and is not intended to limit the scope of the application.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the application belongs. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to limit the application. The reagents and instruments used herein are commercially available, and the characterization means involved can be referred to the related description in the prior art, which will not be described herein.

[0038] In order to further understand the application, the application will be further described in detail below in combination with the best embodiments.

[0039] Example 1

[0040] High-efficiency expression of recombinant Protein AG and application verification experiment of acid and alkali resistance:

[0041] 1、Experimental materials: Protein Maker, Tiangen (Tiangen Biotech (Beijing) Co., Ltd.); Coomassie brilliant blue, GenScript (GenScript Biotech Co., Ltd.); Protein loading buffer, Biosharp (Biosharp Biotech Co., Ltd.); HRP labeled sheep anti-mouse, Biyun Tian (Biyun Tian Biotech Co., Ltd.).

[0042] 2、Experimental scheme:

[0043] The efficient expression of recombinant Protein AG and the application verification method of acid and alkali resistance include the following steps:

[0044] A. Protein AG gene cloning and construction of vector plasmid:

[0045] In order to realize the high acid and alkali resistance of Protein AG and inherit the biological advantages of Protein A and Protein G, the gene of Protein AG is designed and optimized, and the gene SPAC is synthesized by Suzhou Jinyizhi Biotech Co., Ltd. Add 5'(BamHI) and 3'(XhoI), and clone the gene through 5'BamHI and 3'XhoI to the vector pET-28a(+)(Kanamycin), construct the plasmid SPAC in pET-28a(+), prepare 1 mini-scale recombinant plasmid DNA and divide it into 1 tube and 1 tube of glycerol bacteria.

[0046] B. Protein AG protein expression condition optimization:

[0047] In order to realize the optimal expression effect of target protein and the best soluble expression form, the control variable method is used to optimize the induction conditions of prokaryotic expression, and the experimental conditions are changed into two groups, and the induction temperature is unchanged, respectively. Induction time, IPTG concentration.

[0048] C. Protein AG protein expression form analysis and verification:

[0049] In order to realize the expression form of Protein AG protein tends to be soluble, after verifying the expression form analysis, whether to change its expression condition tends to be soluble, so that it does not need to be refolded. Because inclusion body protein only has correct primary amino acid sequence, it cannot form correct spatial three-dimensional structure and does not have its biological activity. Therefore, let the target protein change from inclusion body to soluble expression.

[0050] D: Protein AG purification condition optimization and protein purity and concentration verification:

[0051] In order to realize a large number of purified Protein AG, the unified purification process is realized, so that it can be mass-produced, and therefore the lysis time and elution concentration are optimized in the process of purification, and then the concentration is determined by the protein BCA assay method for subsequent experiments.

[0052] E: Preliminary evaluation of the binding ability of Protein AG and IgG by direct ELISA method:

[0053] The binding ability of Protein AG and IgG can be preliminarily evaluated by enzyme-linked immunosorbent assay (ELISA) experiment, and the intuitive data can show the strength of the binding ability of Protein AG and IgG.

[0054] F: Application research of acid and alkali resistance of Protein AG:

[0055] In order to explore the acid and alkali resistance of Protein AG, two experiments are designed, which are sodium dodecyl sulfate-polyacrylamide gel electrophoresis detection (SDS-PAGE) detection and ELISA detection. The SDS-PAGE detection can directly show whether there is degradation under the gradient experiment of acid and alkali resistance of Protein AG, and the ELISA detection data can show the strength of the binding ability of IgG under the gradient experiment of acid and alkali resistance.

[0056] Example 2

[0057] Protein AG gene cloning and construction of vector plasmid:

[0058] The DNA full sequence synthesis method is used to artificially synthesize the nucleotide sequence and the corresponding amino acid sequence of Protein AG, the nucleotide sequence is SEQ ID NO. 2, and the amino acid sequence is SEQ ID NO. 1. The amino acid sequences of the Ig dominant binding domains of Protein A and Protein G are combined, that is, Protein A+linker+Protein G, the linker sequence is shown as SEQ ID NO. 3; and a cysteine is added at the C terminal, which is designed for single-point connection coupling for fixation, a restriction enzyme site BamHI and XhoI are added to connect with the pET-28a(+) expression vector, the ligation product is transformed into DH5α competent cells, and the plasmid is named SPAC-28a.

[0059] SEQ ID NO. 3: ggcggcggcggcagcggcggcggcggcagcggcggcggcggcagc.

[0060] Example 3

[0061] Optimization of protein expression conditions and analysis of Protein AG expression form verification:

[0062] SPAC-28a (BL21) was taken out of the refrigerator, 5 μl (1:1000) of Kanamycin (100 μg / ml) and 50 μl (1:100) of bacterial solution were taken into a sterile 5 ml test tube medium, and activated by overnight culture at 37°C and 200 r / min until the medium was turbid. The cultured SPAC-28a test tube bacterial solution was transferred into four 5 mL media, 5 μl of Kana (kanamycin) was added, and the culture was carried out at 200 r and 37°C for about 2 h. The OD value was monitored in real time, and the culture was stopped when the OD600 reached 0.6-0.8, and the bottle was taken out. In order to achieve the optimal expression effect and the best soluble expression form of the target protein, the control variable method was used to optimize the induction conditions of prokaryotic expression. The experimental conditions were changed into two groups, and the induction temperature was unchanged. The induction time and the concentration of IPTG were changed. 1, 2, 4, and 5 μl of isopropyl-β-D-thiogalactopyranoside IPTG (1 mmol / L of IPTG prepared in advance) were added to four 5 mL bacterial solutions, respectively. The culture was carried out at 37°C and 200 r / min for 4 h. Samples were taken at 1 h, 2 h, 3 h, and 4 h, respectively. The bacterial precipitate was collected by centrifugation at 8000 rpm and 4°C for 5 min. The supernatant was discarded, and the bacterial precipitate was collected in a centrifuge tube. The protein was resuspended and washed by centrifugation with 1×PBS. The supernatant was discarded. The uninduced bacterial solution was used as a control. 40 μl of all samples were added with 10 μl of 5×protein loading buffer and boiled in boiling water for 10 min. 10 μl of the appropriate sample was taken and identified by 12% SDS-PAGE. An uninduced bacterial solution was used as a control, and SDS-PAGE detection was carried out. From Figure 1 It can be seen that the protein is well expressed at an induction temperature of 37°C for 2, 3, and 4 h, and at an IPTG concentration of 0.4, 0.8, and 1 mmol / L. The protein is named Protein AG. The protein can be induced at a time of 3 h and an inducer concentration of 0.8 mmol / L.

[0063] Example 4

[0064] Analysis and verification of Protein AG expression form:

[0065] SPAC-28a (BL21) was activated and expanded in 500 ml of medium, and the protein was induced under the conditions of an induction temperature of 37°C, an induction time of 3 h, and an inducer concentration of 0.8 mol / L. The supernatant was discarded, and the bacterial precipitate was collected in a centrifuge tube. The protein was resuspended and washed by centrifugation with 1×PBS. The supernatant was discarded. The bacterial precipitate was dissolved with 50 ml of bacterial lysis solution, and the precipitate was broken by ultrasonic ice bath.

[0066] Ice bath break: power 250W over 4s stop 4s break 15-30min to break completely, the bacterial solution is clear and transparent. After breaking, collect into 50ml centrifuge tube and centrifuge 8000rpm for 5-10min, collect the supernatant and the precipitate. Take 40μl of the supernatant and 40μl of the precipitate resuspended with 100μl of 1×PBS, add 10μl of protein loading buffer (5×) and boil in water for 10min, then separate by 12% BT concentration gel electrophoresis. Identify whether the target protein is in the supernatant or the precipitate. Figure 2 It can be seen from the above that there is a clear target band in the supernatant of the broken PA / G protein, and there is a target band in the Protein AG precipitate, but the content of the target protein is much lower than that in the supernatant, and they are all located at 58.5KD, i.e. PA / G is a soluble expression form, and there is no need to induce it at low temperature and change other induction conditions.

[0067] Example 5

[0068] Protein AG purification condition optimization and protein purity and concentration verification:

[0069] First, use pure water to flush A1, A2, B1, and pump at a flow rate of 2ml / min; use eluent (imidazole) to fill B1 pump; use balance liquid (1×PBS pH8.0 Buffer) to balance A2 pipeline; after flushing and balancing, switch A1 loading, 1ml / min, and collect the flow-through when the peak starts to appear; after the flow-through collection is completed, switch the A2 balance pipeline, balance, and then start B1 pump, adjust the gradient to 50mM, 300mM, 500mM gradient elution, 2ml / min, and collect the eluent; after collection is completed, wash A2 pipeline (balance liquid), and use pure water to wash A1 pump, and save the column (20% ethanol aqueous solution). Take 40μl of flow-through, 50mM, 300mM, and 500mM eluent, add 10μl of 5×loading buffer, and boil the sample for 10min for detection. Figure 3 The experimental results show that 50mM imidazole eluent can remove most of the impurity proteins and a small amount of PA / G protein, and 300mM imidazole eluent can elute most of the PA / G protein. The 300mM PA / G protein purified is ultrafiltrated, and after washing with 1×PBS multiple times, the imidazole content is less than 0.2mM, and the salt ion concentration is between 50-100mM. The PA / G protein concentration after ultrafiltration is determined by BCA, and the original solution after ultrafiltration, 3-fold dilution, and 9-fold dilution are used. The protein concentration converted within the known linear relationship is used as the standard, and the obtained protein concentration is 4.22mg / mL (at least >1mg / mL), and the total volume is 5mL. Finally, the protein is stored at -20 degrees to avoid repeated freezing and thawing, and the shelf life is 1 year.

[0070] Example 6

[0071] Direct ELISA method to evaluate the ability of Protein AG and IgG binding:

[0072] Antigen coating: Protein AG protein was diluted to 1 ppm with CBS coating buffer, mixed well. After dilution, add 100 μL / well to the microplate, cover the sealing film, and put it in the 37°C incubator for two hours or 4°C overnight. Take it out and wash it with 1 x washing solution (pH 7.4) 1 time, 250 μL / well, and do not soak and wait. Set up 1 well of blank control (no Protein AG protein coating), 2 wells of negative control (coated with Protein AG protein, but no HRP labeled IgG antibody), 3-6 wells of Protein AG protein coating, 7-8 wells of VP1-28a protein coating, and 9-10 wells of other protein coating.

[0073] Sealing: Add 200 μL / well of RAC blocking solution to the microplate and directly put it in the 37°C incubator for two hours. Take it out and dry it directly.

[0074] Enzyme-labeled secondary antibody binding: Enzyme-labeled antibody HRP labeled goat anti-mouse (IgG) was diluted 5000 times with 1 x PBS, 100 μl / well was added, and it was placed in a constant temperature incubator at 37°C for 30 min. Wash the plate 5 times.

[0075] Color development: Add substrate color developing solution: 100 μl / well of substrate color developing solution was added to each well, and it was placed in a 37°C constant temperature incubator for 15 min.

[0076] Determination: Add 50 μL of stop solution to each well, mix well, and measure the absorbance value at OD450 nm within 5 min.

[0077] Results: From Figure 4 It can be seen that the ability of Protein AG protein to bind with HRP labeled goat anti-mouse (IgG) is significant.

[0078] Example 7

[0079] Application research on the acid and alkali resistance of Protein AG:

[0080] Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) detection: Protein AG was diluted with 0.01 mol / L PBS (pH 7.4) to a final concentration of (2 mg / mL), and 3 volumes of normal saline of different pH values were added. Set pH 2, pH 3, pH 4, pH 5, pH 6, pH 7, pH 8, pH 9, pH 10, pH 11, pH 12, 10 test groups, 4 tubes in each group. Place each sample tube at 37°C in the same pH value group, and sample one tube at 1, 2, 3, and 4 h, respectively. Then detect by SDS-PAGE, whether there are degradation bands. SDS-PAGE result interpretation: from Figure 5 It can be seen that under the action of PBS at each PH value, there is no degradation band, indicating that Protein AG is more resistant to acid and alkali in a short time.

[0081] ELISA detection: Protein AG was diluted with 0.01 mol / L PBS (pH 7.4) to a final concentration of 1 ppm. Coat 100 μL of Protein AG overnight at 4°C on an ELISA plate, wash 5 times with PBST, then seal the plate with 200 μL of BSA blocking solution overnight at 4°C, wash 5 times, then add 200 μL of PBS corresponding to the degradation of pH 3-pH 12, incubate for 1.5 h, 2 replicate wells for each sample. Set normal control (without PBS), blank control (normal blocking without secondary antibody), and negative control (normal blocking with secondary antibody). Wash 5 times, then add HPR labeled goat anti-mouse 2 antibody (1:5000) 100 μL directly to each well, develop for 10 min, and immediately terminate with 50 μL of termination solution and read the value on the enzyme label.

[0082] Direct ELSIA experimental result interpretation: from Figure 6 It can be seen that the average OD values of each PH value sample measured by PBS fluctuate little, and the standard deviation of the OD values of PH 3-12 is only 0.188, among which the standard deviation of the sample with PH value 7 is the smallest, 0.022, and the standard deviation of the sample with PH value 11 is the largest, 0.197, indicating that the data difference of Protein AG ELSIA experiment under the action of PBS at each PH value is not large, further indicating that Protein AG can tolerate in the range of pH 3-pH 12.

[0083] The OD values under the influence of PBS at each pH value are compared with the OD values obtained under normal experiment, and PBS at each pH value fails to affect the binding of Portein AG and goat anti-mouse 2 antibody.

[0084] Conclusion: The fusion protein Protein AG can tolerate in the range of pH 3-pH 12, and does not reduce the binding ability with immunoglobulin.

[0085] The prior art document (CN102676562A) mentioned in the background section of the present technology is quite different. The preparation method of the present patent technology prepares a fusion protein by gene synthesis, but the difference is that in the present technology, the sequence after fusion is also modified and optimized in advance, including but not limited to: optimization of the 5' region (translation initiation efficiency), relative synonymous codon usage (RSCU), DNA repeat sequence, mRNA secondary structure, GC content, SD sequence, exclusion of specified restriction enzyme sites, etc. These modifications and optimizations can increase the codon adaptation index (CAI) and GC content of the gene sequence. CAI is the adaptation coefficient of the gene, which uses the optimal codon for all codons of the gene encoding the protein. The increase in GC content makes the DNA more dense and less likely to denature under heat or alkaline conditions. That is, the translated protein of the gene sequence can adapt to the properties of polar substances, expand the pH range of the buffer used, and have a broader application prospect and range.

[0086] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modifications, equivalent replacements or improvements made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A fusion protein PA-G, characterized in that, The fusion protein PA-G fuses the Ig dominant binding domain of protein A and protein G, and the amino acid sequence of the fusion protein PA-G is the sequence shown in SEQ ID NO.

1.

2. A gene encoding the fusion protein PA-G of claim 1, characterized in that, The nucleotide sequence of the gene is the sequence shown in SEQ ID NO.

2.

3. A recombinant expression vector, characterized in that, The recombinant expression vector is inserted with the gene according to claim 2.

4. The recombinant expression vector according to claim 3, wherein, The expression vector is a pET series vector, and the pET series vector is a pET-32a expression plasmid, a pET-26b expression plasmid or a pET-28a expression plasmid.

5. A recombinant strain expressing a recombinant fusion protein PA-G, characterized in that, The recombinant strain is obtained by transforming the E. coli competent cell with the recombinant expression vector according to claim 3 or 4.

6. The recombinant bacterial strain of claim 5, wherein the recombinant expression fusion protein PA-G is represented by the following formula: ###0001### wherein, X is a linker; and Y is a protein. The E. coli competent cell is an E. coli BL21 competent cell, an E. coli rosetta competent cell or an E. coli DH5α competent cell.

7. A method of preparing the fusion protein PA-G according to claim 1, characterized in that, The method is a fermentation method for preparing the fusion protein PA-G according to claim 1 by using the recombinant strain according to claim 5 or 6.

8. The method of claim 7, wherein, The method comprises the following steps: constructing a recombinant expression vector containing the gene according to claim 2 by using the expression vector pET-28a; transforming the E. coli DH5α competent cell with the recombinant expression vector, and obtaining a recombinant strain capable of expressing the fusion protein PA-G according to claim 1 by culturing and screening; after culturing the recombinant strain, inducing protein expression by IPTG, and separating and purifying the expressed fusion protein PA-G.

9. The fusion protein PA-G according to claim 1 is applied in antibody purification or immunoprecipitation.

10. The gene according to claim 2, the recombinant expression vector according to claim 3 or 4, or the recombinant strain according to claim 5 or 6 is applied in antibody purification or immunoprecipitation.

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