Recombinant streptavidin, its encoding gene, recombinant plasmid and genetically engineered bacteria and their application

Abstract

The present invention discloses a recombinant streptavidin, a coding gene, a recombinant plasmid and a genetic engineering bacterium and an application thereof. The amino acid sequence of the recombinant streptavidin is shown in SEQ ID NO.1, and the coding gene sequence thereof is shown in SEQ ID NO.5. The recombinant streptavidin is successfully expressed in Escherichia coli by gene recombination technology after sequence modification and addition of a polypeptide. The streptavidin has good specificity and high titer. The recombinant streptavidin is applied as an amplification system to a colloidal gold antigen detection kit, has the advantages of high specificity, high sensitivity and good stability, meets the requirements of a colloidal gold chromatography detection kit, and can be applied as an amplification system to a chromatography detection reagent.

Description

Technical Field

[0001] The present invention relates to the field of gene recombination and immune diagnosis technology, and in particular to a recombinant streptavidin, its encoding gene, recombinant plasmid and genetic engineering bacteria and applications thereof. Background Art

[0002] Since the 1970s, with the development of immunochemical technology, various biotin labeling technologies have been gradually established, and the biotin-avidin system has been used as an amplification system in various fields of immunology. Subsequently, streptavidin (SA) with similar biological properties to avidin (AV) was discovered, and when biotin-streptavidin is used to detect antigen-antibody systems, its nonspecific binding is much lower than that of biotin-avidin, and its sensitivity is higher. Therefore, the biotin-streptavidin amplification system is increasingly widely used in various fields of immunology.

[0003] Streptavidin (SA) is a protein secreted by Streptmyces avidinii, which has a highly specific affinity for biotin. Streptavidin is a tetramer, and each subunit can bind to one molecule of biotin on average. The affinity between Streptavidin and Biotin is very strong. During the secretion process, the natural SA removes the N-terminal 24AA signal peptide. The monomer consists of 169 amino acids, with a molecular weight of 16.5kDa, and the tetramer molecular weight is 66.0kDa. However, the molecular weight of the separated and purified SA is about 60kDa, and the molecular weight of each single chain is about 15kDa. Studies have shown that the full-length protein is easily cleaved by proteases secreted from cells during the exocrine fermentation of streptavidin bacteria, removing 10-12 amino acid residues at the N-terminus and 19-21 amino acids at the C-terminus to become incomplete SA, so the separated and purified SA will be shorter than the full-length protein; studies have shown that the sequences at both ends of the SA amino acid are hydrophobic sequences, which will reduce the solubility of the protein. At the same time, studies have confirmed that the sequences at both ends of SA are also not conducive to the binding of SA to biotin. During the secretion process of SA, proteases remove these two sequences, restore the solubility of SA, greatly reduce the cytotoxicity caused by excessive binding to biotin in the cell, and play a role in protecting cells. Truncated SA, called core streptavidin (Core Streptavidin, CSA), has good solubility, biological activity, stability, acid resistance, urea resistance and high guanidine hydrochloride capacity. It is the main form of streptavidin currently used in China.

[0004] At present, streptavidin is widely used in coating microspheres, 96-well plates for enzyme-linked immunosorbent assay, labeling various enzymes, fluorescein, etc. However, for colloidal gold chromatography detection reagents, the SA products of most manufacturers have poor stability, and are prone to problems such as thick bands, blurring, light color development, and poor specificity when used. Therefore, it is of great significance to develop efficient recombinant streptavidin that is more suitable for use on the colloidal gold platform.

[0005] Therefore, the prior art needs to be further improved. Summary of the invention

[0006] In view of the above problems, the present invention provides a new recombinant streptavidin, its encoding gene, recombinant plasmid and genetically engineered bacteria, and also provides a preparation method and application of the recombinant streptavidin. The activity and stability of the improved recombinant streptavidin are significantly improved, and it is suitable for the application field of colloidal gold chromatography detection reagents.

[0007] The technical solution provided by the present invention is specifically as follows:

[0008] In a first aspect, the present invention provides a recombinant streptavidin, whose amino acid sequence is shown in SEQ ID NO.1. The sequence of the recombinant streptavidin (named as rSA (Recombinant Streptavidin)) is as follows: MAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGHYESAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAASGGGS AEAAACDKTSSSHTCPKYLKKYIAWPLQGWQATFGG GDHPPP

[0009] Among the above sequences, part of the sequence selected the amino acid sequence of 13-139 in the core region of streptavidin as shown in SEQ ID NO.2, and the RYD of the amino acids at positions 59-61 in the sequence was mutated to HYE. Since the RYD sequence can bind to cells with fibronectin receptors, after this mutation, the non-specific binding of the protein to tissue cells can be effectively avoided.

[0010] The sequence of SEQ ID NO.2 is:

[0011] AEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS

[0012] Secondly, the carboxyl end of the core region of streptavidin is connected to a polypeptide fragment (sequence shown in SEQ ID NO.3) through a connecting peptide (sequence GGGS, as shown in SEQ ID NO.4). The polypeptide fragment helps to improve the renaturation rate of streptavidin and improve the potency of recombinant streptavidin in chromatographic detection.

[0013] The sequence of SEQ ID NO.3 is:

[0014] AEAAACDKTSSSHTCPKYLKKYIAWPLQGWQATFGGGDHPPPK

[0015] Compared with the existing core streptavidin (CSA), the recombinant streptavidin rSA obtained after the above sequence improvement has a higher titer, and its stability and specificity are significantly improved. When used in colloidal gold test strips, the strip color is clear and there is no blurring. Its sensitivity reaches 5pg / ml, which is much higher than the CSA in the control group. In the specific detection experiment, the recombinant streptavidin rSA of the present application has high specificity and high result accuracy. No blocker needs to be added in the application, which saves costs. False positives in the control group need to be eliminated by adding additional blockers.

[0016] In a second aspect, the present invention provides a recombinant streptavidin, the sequence of which carries the amino acids LE and a His tag: HHHHHH on the carrier at the carboxyl end of the aforementioned amino acid sequence.

[0017] By adding the His tag, the recombinant streptavidin bacteria can be easily purified by metal chelate chromatography (IMAC), thereby improving the purification efficiency of the protein without affecting the activity of the protein.

[0018] Therefore, in other embodiments, if the recombinant streptavidin is not purified by metal chelate chromatography, the His tag can be omitted.

[0019] In a third aspect, the present invention also provides a coding gene for the recombinant streptavidin. The coding gene can be obtained according to the amino acid sequence of the recombinant streptavidin provided above. The coding gene can also be obtained according to the codon preference of the expression system used. Therefore, the coding gene for the recombinant streptavidin to be protected by the present invention is not limited to the gene sequence shown in SEQ ID NO.5.

[0020] Preferably, the present invention provides a gene encoding the recombinant streptavidin, the sequence of which is shown in SEQ ID NO. 5. The sequence is a preferred sequence after codon optimization based on the codon preference of E. coli, and has high yield, high activity and good stability in the E. coli expression system.

[0021] In a fourth aspect, the present invention provides a recombinant vector, wherein the recombinant expression vector is formed by the recombination of an expression vector and the aforementioned recombinant streptavidin encoding gene.

[0022] The expression vector is a pET series expression vector, such as pET-28ap, pET-24a or pET-32a plasmid.

[0023] In a fifth aspect, the present invention further provides a recombinant engineered bacterium, wherein the recombinant engineered bacterium is formed by transforming the aforementioned recombinant expression vector into a host bacterium.

[0024] Preferably, the host bacteria is Escherichia coli, such as Escherichia coli BL21 (DE3), Escherichia coli Rosseta or other mutant expression strains of Escherichia coli.

[0025] In a sixth aspect, the present invention further provides a method for preparing the above-mentioned recombinant streptavidin, characterized in that it comprises the following steps:

[0026] 1) Construction of a recombinant expression vector: amplifying the aforementioned recombinant streptavidin coding gene, performing enzyme digestion, and then connecting the coding gene with the expression vector subjected to the same enzyme digestion to obtain a recombinant expression vector;

[0027] 2) Construction of recombinant engineered bacteria: transforming the recombinant expression vector into the host bacteria and screening to obtain the recombinant engineered bacteria;

[0028] 3) Extraction and purification of recombinant streptavidin: The recombinant engineered bacteria are fermented, and the recombinant streptavidin is induced to express, and the expression product, recombinant streptavidin, is extracted and purified.

[0029] In step 1), primers with sequences as shown in SEQ ID NO.6 and SEQ ID NO.7 are used for amplification.

[0030] Optionally, the expression vector is a pET series expression vector, and the host bacteria is Escherichia coli.

[0031] Alternatively, the recombinant streptavidin can be separated and purified by methods such as Ni ion affinity chromatography.

[0032] Preferably, the extraction and purification method of recombinant streptavidin is:

[0033] The recombinant engineered bacteria are fermented, and then an inducer is added to continue culturing. After the cultivation is completed, the bacterial precipitate is collected by centrifugation, the bacterial precipitate is crushed and centrifuged, the inclusion body is collected and redissolved, and finally purified by a nickel ion affinity column. After gradient renaturation dialysis, recombinant streptavidin rSA is obtained.

[0034] In a seventh aspect, the present invention further provides a kit comprising the aforementioned recombinant streptavidin. The kit may be any existing detection kit, such as a novel coronavirus antigen detection kit.

[0035] In an eighth aspect, the present invention also provides the use of the above-mentioned recombinant streptavidin in colloidal gold chromatography detection kit products, such as the use of novel coronavirus antigen detection kits, hepatitis B detection kits, malarial parasite detection kits, mycoplasma pneumoniae detection kits, and chlamydia pneumoniae detection kits. Since recombinant streptavidin is versatile in various kits, it is widely used.

[0036] The present invention has the following beneficial effects:

[0037] 1. The recombinant streptavidin provided by the present invention is successfully expressed in Escherichia coli through gene recombination technology after sequence modification and the addition of a polypeptide. The recombinant protein has the advantages of good specificity and high titer. The recombinant streptavidin is used as an amplification system in a colloidal gold antigen detection kit, which has the advantages of high specificity, high sensitivity and good stability, and meets the requirements of a colloidal gold chromatography detection kit.

[0038] 2. The recombinant streptavidin of the present invention has the advantages of high yield, high titer, excellent specificity, good product applicability, etc. in the E. coli expression system. The recombinant streptavidin can be applied to chromatography detection reagents as an amplification system on a colloidal gold platform, and has the advantages of high titer, excellent specificity, good stability, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The results of SDS-PAGE and HPLC detection of recombinant streptavidin;

[0040] Figure 2 This is the result of ELISA testing the titer of recombinant streptavidin;

[0041] Figure 3 To detect the optimal coating concentration of rSA;

[0042] Figure 4 To detect the optimal coating concentration of cS;

[0043] Figure 5 The results of the rSA thermal stability test; the left test strips in each group were stored at room temperature, and the right test strips were accelerated at 55°C. DETAILED DESCRIPTION

[0044] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are conventional methods in the art unless otherwise specified.

[0045] The various molecular biology enzymes used in the present invention were purchased from Dalian Bao Bioengineering Co., Ltd.

[0046] Example 1 Construction of recombinant plasmid pET24a-rSA and construction of expression strain

[0047] 1. Experimental methods

[0048] (1) Design and amplification of recombinant streptavidin sequences

[0049] A. Referring to the amino acid sequence of WP_229920816.1 in the GenBank database, the sequence of the N-terminal signal peptide at positions 1-24 of the sequence was removed, and the 13-139aa fragment thereof (as shown in SEQ ID NO.2) was selected, and then the three amino acids RYD at positions 59-61 were mutated to HYE, i.e., the selected streptavidin amino acid sequence;

[0050] At the same time, the carboxyl end of the sequence is connected to the polypeptide sequence shown in SEQ ID NO.4 through a connecting peptide (such as the sequence shown in SEQ ID NO.3: GGGS) to obtain the recombinant streptavidin amino acid sequence shown in SEQ ID NO.1; after being connected to the vector, the carboxyl end of the sequence carries an enzyme cleavage site, translated amino acids LE and a 6H tag (HHHHHH), and the recombinant streptavidin amino acid sequence is further obtained.

[0051] B. Gene synthesis:

[0052] The above amino acid sequence was codon optimized to obtain the coding gene rSA of the recombinant streptavidin sequence, the sequence of which is shown in SEQ ID NO.5, and Beijing Qingke Biotechnology Co., Ltd. was arranged to synthesize it;

[0053] C. Primer design and amplification of target gene:

[0054] ① Using the synthesized coding gene rSA as a template, use primer5 primer design software to design amplification primers P1 and P2. According to the primer design and restriction site design principles, combined with the pET24a vector restriction site map, add restriction sites and protection bases in front of the upstream and downstream primers respectively, and use primers P1 and P2 to amplify the nucleic acid coding sequence SEQ ID NO.8 of rSA.

[0055] Design primers as follows:

[0056] P1: 5'-GGAATTC CATATG GCTGAAGCAGGGATAACAGGA-3'(Nde I)P2: 5'-CCCTCGAGTTTTGGAGGTGGATGGTCGCCA-3'(Xho I)

[0057] Primer P1 is shown in the sequence listing as SEQ ID NO.6, primer P2 is shown in the sequence listing as SEQ ID NO.7, and the amplified sequence is shown in SEQ ID NO.8.

[0058] ② Add the reaction system according to Table 1 and perform gene amplification according to the amplification program in Table 2.

[0059] Table 1 PCR reaction system

[0060]

[0061] Table 2 Amplification procedures

[0062]

[0063] (2) Construction of recombinant expression strain

[0064] After the above PCR amplification product is recovered, it is double-digested with Nde I and Xho I, and the digestion product is recovered and connected to the vector pET24a double-digested with Nde I and Xho I. After transformation to DH5α and PCR identification of the recombinant plasmid, a positive transformant is obtained, and the recombinant plasmid is extracted from the positive transformant, and the recombinant plasmid is transformed into the Escherichia coli expression strain BL21 to obtain the recombinant protein expression strain: B / pET24a-rSA.

[0065] At the same time, a control recombinant core streptavidin (cSA) was prepared, whose amino acid sequence was selected from streptavidin 13-139aa, and the amino acid sequence was shown in SEQ ID NO. 9. The coding sequence of the recombinant core streptavidin (cSA) was amplified according to the aforementioned method, and the corresponding recombinant plasmid was prepared using it, and the recombinant expression strain was prepared by transformation for subsequent experiments.

[0066] Example 2: Recombinant protein expression and purification

[0067] 1. Induced expression of protein:

[0068] 10 μl of the bacterial solution of the recombinant plasmid expression strain was inoculated into 5 ml LB medium containing 100 ug / ml kanamycin (Kan) (purchased from Solebol), and cultured at 37°C, 200 rpm overnight (about 16 h). 200 μl of the bacterial solution was added to 60 ml LB medium containing the same concentration of Kan, and cultured at 37°C, 200 rpm overnight. All the bacterial solution was inoculated into 1 L LB medium containing the same concentration of Kan, and cultured at 28°C, 150 rpm for 2-2.5 h, and the OD was measured. 600 At a concentration between 0.5 and 0.8, add the inducer IPTG (purchased from AMRESCO) with a final concentration of 0.5 mM, continue to culture at 28°C, 150 rpm with shaking for 4.5 h, then centrifuge at 6000 rpm for 5 min to collect the bacterial precipitate; resuspend the bacterial precipitate with 50 ml 20 mM PBS (pH 7.4), centrifuge at 6000 rpm for 15 min, and collect the precipitate.

[0069] 2. High pressure crushing:

[0070] The bacterial precipitate was resuspended with 30 ml of 20 mM PBS (pH 7.4) and subjected to low-temperature high-pressure crushing. The parameters were set as follows: pressure value 910-980 bar, 3 cycles, temperature setting 4°C, and the actual operating temperature should not be higher than 13°C. After the crushing, the crushed product was centrifuged at 9000 rpm for 30 minutes at low temperature, the precipitate was collected, and then re-dissolved with 8 M urea overnight. The re-dissolved sample was centrifuged at 10000 rpm for 40 minutes at low temperature, and the upper supernatant was collected to obtain a protein sample.

[0071] 3. Protein purification:

[0072] Take the Bestarose-FF chromatography column of IMAC (purchased from Boglon) for affinity chromatography. The specific process is as follows:

[0073] The chromatography column was balanced with 10 column volumes of ultrapure water; the column was balanced with 10 column volumes of equilibration buffer (20mM PB+8M urea, pH7.4) and then the protein sample obtained in the previous step was added; after the loading was completed, the unbound protein was washed away with 10 column volumes of equilibration buffer; finally, the target protein was eluted with elution buffer (20mM PB+8M urea+100m M imidazole, pH7.4) to obtain an eluted sample. The eluted sample was renatured according to the gradient of 4M Urea (urea) → 2M Urea → 0M Urea, and the recombinant protein was obtained by concentration after renaturation.

[0074] 4. Detection of purified protein

[0075] The purified protein was tested by SDS-PAGE and SEC-HPLC (size exclusion high performance liquid chromatography). Figure 1 As shown:

[0076] (1) Protein electrophoresis showed that the size of the recombinant streptavidin rSA monomer was consistent with the theoretical value of 19.3 kDa.

[0077] (2) In the SEC-HPLC test results, by calculating the peak area, the purity of rSA was 94.7%, and there was no obvious aggregate, and the homogeneity was excellent. The yield of rSA was analyzed and its yield was as high as 1g / L, which can be mass-produced.

[0078] Example 3 ELISA to detect the titer of recombinant streptavidin

[0079] 1. Experimental methods

[0080] Recombinant streptavidin rSA and control recombinant core streptavidin cSA were coated at 4°C overnight in a gradient of 0.25ug, 1ug, and 4ug, respectively. The plates were washed with PBST buffer, blocked with 2% BSA at 37°C for 1h, and then washed. The primary antibody biotinylated mouse anti-human IgG monoclonal antibody (Shandong Shuojing Biology) was added and incubated at 37°C for 1h. The plates were washed, and the secondary antibody sheep anti-mouse-HRP (Shandong Shuojing Biology) was added and incubated at 37°C for 30min. The TMB method was used for color development, and then OD was measured. 450 reading.

[0081] 2. Experimental results and analysis

[0082] The results are as follows Figure 2 As shown, under three different concentration conditions, the titer of recombinant streptavidin rSA was significantly higher than that of the recombinant core streptavidin cSA in the control group.

[0083] Example 4 Application of recombinant streptavidin in novel coronavirus antigen detection test strips 1. Test strip preparation

[0084] (1) Colloidal gold labeling

[0085] Take 1mL of colloidal gold and put it in a small glass cup, stir and add an appropriate amount of 0.2M K2CO3 to adjust the pH to 7.6, stir for 1min; add 20μg monoclonal antibody 1 (new coronavirus N protein antibody 1, Shandong Shuojing Biology), stir for 1min; add 100μL of 1% PEG20000, stir for 1min; centrifuge at 5000g for 10 minutes, discard the supernatant, and resuspend the precipitate with 100ul colloidal gold diluent (20mM PB, 150mM NaCl, 0.1% PEG20000, 0.1% TritonX-100, 2% Sucrose, 0.01% NaN3); mix thoroughly and store at 4°C. The above gold-labeled complex was diluted 10 times with colloidal gold diluent and evenly applied to the glass fiber, dried at 37°C to make a gold-labeled pad.

[0086] (2) Biotinylated monoclonal antibody 2

[0087] The monoclonal antibody 2 (novel coronavirus N protein antibody 2, Shandong Shuojing Bio) to be biotinylated was dialyzed into phosphate buffer and the protein molarity was calculated; the biotin was balanced to room temperature. 2 mg Sulfo-NHS-Biotin was added to 100 ul ultrapure water and mixed with monoclonal antibody 2 at a ratio of 12-20 times at room temperature for 30 minutes. The unbound free biotin was removed by dialysis.

[0088] Dilute the biotinylated monoclonal antibody 2 and evenly apply it on the glass fiber, dry it at 37℃ to make S pad. (3) Nitrocellulose membrane (NC membrane) coating

[0089] The prepared streptavidin recombinant protein was diluted with coating solution (20mM PBS) to the working concentration as the test line (T line) coating material, and gradient coating was performed at 0.5mg / ml, 1.0mg / ml, and 1.5mg / ml respectively. A film drawing instrument was used to draw lines on the NC membrane, and then baked at 37°C for 30min to complete the solid phase of the raw material.

[0090] (4) Assembly

[0091] Assemble the solid-phased NC membrane with the sample pad, S pad, gold label pad, PVC base plate, absorbent paper and Mark sticker, and cut them into 3-4 mm test strips with a strip cutter for later use.

[0092] 2. Performance testing

[0093] (1) Optimal coating concentration and titer detection

[0094] ① Experimental methods:

[0095] The test strips prepared above were used to test the novel coronavirus N protein control product (Qingdao Shuojing). The control product concentrations were graded and diluted at 1ug / ml, 10ng / ml, 1ng / ml, 100pg / ml, 50pg / ml, 10pg / ml, and 5pg / ml. The rSA test results were as follows: Figure 3 , cSA test results are as follows Figure 4 The absorbent paper portion of the test strip 1 is marked with a line. The test strip is marked with 1, 2, and 3 bars from left to right, indicating that the rSA coating concentrations are 0.5 mg / ml, 1.0 mg / ml, and 1.5 mg / ml, respectively.

[0096] ② Experimental results and analysis

[0097] like Figure 3 The results show that by comparing the depth of color development at different coating concentrations, it can be seen that through the detection of quality control products, the color comparison shows that the optimal coating concentration of rSA is 1.5mg / ml. Under this coating condition, the color development is the darkest and the color line is uniform.

[0098] Figure 4 The control group is cSA. The color comparison shows that the best coating of cSA is 1.0 mg / ml, and the higher the coating concentration of the control group, the lighter the color, and a hook effect occurs. This shows that the linear range of the control group is narrow, and the control group fails to improve the color effect as the coating concentration increases. As the coating concentration increases, the line becomes thicker and there is a blurring phenomenon (this situation shows that the titer of cSA is low. When the coating amount increases, the membrane cannot carry so much protein and blurring is likely to occur).

[0099] contrast Figure 3 and Figure 4 It can be seen that at the optimal coating concentration, the color development of rSA is significantly better than that of the control group cSA.

[0100] (2) Sensitivity test

[0101] ① Experimental methods:

[0102] rSA and control cSA were coated at the optimal coating concentrations, respectively, and prepared into test strips for detecting N protein controls: 1ug / ml, 10ng / ml, 1ng / ml, 100pg / ml, 50pg / ml, 10pg / ml, 5pg / ml, and negative control.

[0103] ② Experimental results and analysis

[0104] The results are shown in Table 3. The color development of rSA is significantly stronger than that of the control. When detecting 10pg / ml, rSA can still be detected. When detecting 5pg / ml quality control, the band color is light but still discernible, which is at the critical value. It can be determined that the minimum detection concentration of rSA is 5pg / ml. The minimum detection concentration of the control group is 50pg / ml. The color development of 10pg / ml and 5pg / ml quality control products is negative, and no target bands can be observed. This shows that the sensitivity of rSA is much higher than that of the control cSA.

[0105] The minimum detection concentration of rSA was 5pg / ml; the minimum detection concentration of the control group was 50pg / ml, which indicated that the sensitivity of rSA was much higher than that of the control cSA.

[0106] Table 3 Quality control product test results

[0107] N Protein Control Concentration rSA test results cSA test results 1ug / ml, ++++ ++++ 10ng / ml ++++ +++ 1ng / ml +++ ++ 100pg / ml ++ + 50pg / ml + + 10pg / ml + - 5pg / ml + - Negative control - -

[0108] Note: + indicates positive, - indicates negative, +- indicates critical value, and the more + the darker the color.

[0109] (3) Specificity detection

[0110] The colloidal gold test strip prepared in the present application was used to test 100 negative volunteer throat swab samples. The test results showed:

[0111] ① The test strips coated with rSA were all negative for these 100 samples, showing high specificity and accurate test results.

[0112] ② In the control group (test paper coated with cSA), there were 2 false positives and 1 background. This problem could be eliminated by adding additional blockers. This shows that rSA has better specificity than the control cSA, and no blockers need to be added during its application, further saving detection costs.

[0113] (4) Product thermal stability

[0114] ① Experimental methods:

[0115] The colloidal gold test strips prepared by coating recombinant streptavidin were subjected to accelerated destruction at 55°C for 7 days. At the same time, an experimental control group was set up and the control group was treated at room temperature for 7 days. After 7 days, the colloidal gold test strips of the experimental group and the control group were tested against the quality control products, and the concentrations of the quality control products were 1μg / ml, 100ng / ml, 10ng / ml, 1ng / ml, and 100pg / ml.

[0116] ② Experimental results and analysis:

[0117] Test results such as Figure 5As shown, compared with the control group treated at room temperature, there is no difference in the color development results of the test paper of the experimental group accelerated at 55°C for 7 days for detecting the quality control product, which indicates that the recombinant streptavidin of the present application has good thermal stability.

[0118] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.

Claims

1. A recombinant streptavidin, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

2. A recombinant streptavidin, characterized in that: It is obtained by adding LE and His tag: HHHHHH to the carboxyl terminus of the amino acid sequence as claimed in claim 1.

3. The recombinant streptavidin encoding gene according to claim 1, characterized in that: The sequence is shown as SEQ ID NO.

5.

4. A recombinant expression vector, characterized in that: The recombinant expression vector is formed by the recombination of an expression vector and the coding gene of the recombinant streptavidin according to claim 1 or 2 or the coding gene according to claim 3.

5. The recombinant expression vector according to claim 4, characterized in that The expression vector is a pET series expression vector.

6. A recombinant engineered bacterium, characterized in that: The recombinant engineered bacteria are formed by transforming the recombinant expression vector as claimed in claim 5 into a host bacterium.

7. The recombinant engineered bacterium according to claim 6, characterized in that The host bacteria is Escherichia coli.

8. A method for preparing recombinant streptavidin as claimed in claim 1, characterized in that: The following steps are involved: The recombinant engineered bacteria as described in claim 6 are cultured, an inducer is added and the culture is continued, and after the culture is completed, the bacterial precipitate is collected by centrifugation, the bacterial precipitate is crushed by a high-pressure homogenizer, centrifuged, the inclusion body is redissolved, and the nickel ion affinity column is purified, and the recombinant streptavidin is obtained after gradient refolding and dialysis.

9. A detection kit, characterized in that: It comprises the recombinant streptavidin as claimed in claim 1.

10. Use of the recombinant streptavidin according to claim 1 or 2 in the preparation of a colloidal gold chromatography detection kit.

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