Recombinant protein for detecting leishmania as well as coding gene, preparation method and application thereof

Abstract

The invention discloses a recombinant protein for detecting leishmania as well as a coding gene, a preparation method and application of the recombinant protein, an amino acid sequence of the recombinant protein is formed by fusing a K39 protein partial sequence, a K26 protein partial sequence and a K9 protein complete sequence, and the amino acid sequence of the recombinant protein is as shown in SEQ ID NO.1. The recombinant protein used in the invention is fused with a partial sequence of a K39 protein, a partial sequence of a K26 protein and a complete sequence of a K9 protein; the recombinant protein is combined with a double-antigen sandwich enzyme-linked immunosorbent assay and an optical excitation chemiluminescence technology, a clinical detection technology which is high in sensitivity, short in time consumption and simple and convenient to operate is established, and the technical problem that high sensitivity and high detection efficiency of commercialized products for detecting the canine leishmanian antibody cannot be considered at the same time at present is solved.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and in particular relates to a recombinant protein for detecting Leishmania and a coding gene thereof, a preparation method and an application thereof. Background Art

[0002] Leishmaniasis is a zoonosis caused by Leishmania, and the World Health Organization has identified leishmaniasis as an important public health problem. Leishmaniasis includes asymptomatic infection and three main clinical syndromes. In the dermis, the parasite causes symptoms in the form of skin lesions, which can be localized or diffuse; in the mucosa, Leishmania causes symptoms in the form of mucosal lesions; infection metastasis to the spleen and liver can cause visceral damage, causing visceral leishmaniasis (also known as kala-azar). The parasite can also enter other organs, such as lymph nodes, bone marrow and lungs, and in rare cases, can even reach the brain. If not treated appropriately and promptly, most patients die from complications of other diseases within 1 to 2 years after onset. Globally, cutaneous leishmaniasis is the most common form, but visceral leishmaniasis is the main prevalent in my country.

[0003] Dogs are the main intermediate hosts of Leishmania. Since there is no effective vaccine for the disease, there are no relevant treatment drugs for dogs after infection, and the mortality rate is as high as 95%, eliminating sick dogs and asymptomatic infected dogs is the priority strategy for preventing and controlling kala-azar, especially the early diagnosis of asymptomatic infected dogs is the most critical, so rapid and accurate diagnosis methods are the main means to curb the spread of the disease.

[0004] In the existing technology, the detection technology of canine Leishmania (canis Leishmania) is mainly divided into two categories: pathogen diagnosis based on PCR technology and serological experiments based on immune detection technology. However, there are no relevant detection products in China, and we can only rely entirely on expensive imported detection kits, which makes it difficult to conduct large-scale group detection in China. In addition, the current detection methods have certain defects, such as indirect ELISA, which is cumbersome, time-consuming, and low in detection efficiency; colloidal gold detection products have low sensitivity and specificity.

[0005] Diagnostic antigens are the core reagents for detecting canine Leishmania antibodies. From the earliest direct agglutination test to indirect immunofluorescence and then to the current mainstream lateral flow immunochromatography test and ELISA test, diagnostic antigens have evolved from crude parasites to single K39 proteins and then to fusion-expressed recombinant proteins; K39 is the diagnostic antigen recommended by WOAH, with high sensitivity and specificity. At present, the diagnostic antigen of immunoassay products in actual applications is still K39, and my country does not have its own immunodiagnostic products. In particular, in the recent outbreak of canine Leishmaniasis in Yangquan, Shanxi, all detection cards were imported. Summary of the invention

[0006] In view of the shortcomings of existing detection methods such as "low sensitivity, long detection time, complex experimental operation, and unsuitability for routine clinical detection", the present invention provides a recombinant protein for detecting Leishmania and its encoding gene, preparation method and application. The protein can be used for the preparation of homogeneous chemiluminescence detection reagent. The detection method has high sensitivity, good specificity, strong stability, simple operation, short detection time, and is suitable for routine clinical detection of Leishmania.

[0007] In order to achieve the above object, the present invention provides the following solutions:

[0008] In a first aspect, the present invention provides a recombinant protein for detecting Leishmania, wherein the amino acid sequence of the recombinant protein is a fusion of a partial sequence of a K39 protein, a partial sequence of a K26 protein and the entire sequence of a K9 protein, and the amino acid sequence of the recombinant protein is shown in SEQ ID NO.1.

[0009] In a second aspect, the present invention provides a gene encoding a recombinant protein of Leishmania, wherein the gene encoding is recombined from a partial sequence of K39 nucleotides, a partial sequence of K26 nucleotides and a complete sequence of K9 nucleotides, and the nucleotide sequence of the gene encoding is shown in SEQ ID NO.2.

[0010] The coding gene is to express the recombinant protein in Escherichia coli. The codons are optimized according to the codon preference of Escherichia coli. Different species have different usage frequencies of synonymous codons, and this codon preference has an impact on the translation process. If an mRNA has many clustered rare codons, this will have a negative impact on the movement speed of the ribosome and greatly reduce the protein expression level. Here, the coding gene is codon optimized for Escherichia coli expression to improve protein expression efficiency.

[0011] In a third aspect, the present invention provides an expression vector comprising the encoding gene.

[0012] In a preferred embodiment, the expression vector is pET-28a, which has kanamycin resistance, and the expressed recombinant protein has a histidine (His) tag.

[0013] In a fourth aspect, the present invention provides a host cell, comprising the expression vector.

[0014] In a preferred embodiment, the host cell is a eukaryotic host cell or a prokaryotic host cell.

[0015] Furthermore, the host cell is a prokaryotic host cell. The host cell is preferably Escherichia coli; more preferably, the Escherichia coli is BL21. Using Escherichia coli for expression has the advantages of short cycle, low cost, and high expression amount.

[0016] In a fifth aspect, the present invention provides a method for preparing a recombinant protein for detecting Leishmania, comprising the step of inducing the host cell to express the protein.

[0017] In order to make the recombinant protein better maintain the active site, the present invention selects relatively mild culture and induction conditions. The induction temperature during expression is 20-35°C, the induction speed is 100-300rpm, and the induced IPTG concentration is 0.5-2mM. This allows the recombinant protein to be expressed more slowly and has sufficient time to form a spatial conformation.

[0018] There are many ways to purify recombinant proteins, such as ion exchange chromatography, gel filtration chromatography, affinity chromatography, etc. The present invention selects the affinity chromatography method, because the His tag is added to the recombinant protein, a one-step purification can achieve a higher purity.

[0019] In a sixth aspect, the present invention provides use of the recombinant protein for detecting Leishmania in preparing a kit.

[0020] Compared with the prior art, the beneficial technical effects of the present invention are:

[0021] The recombinant protein used in the present invention fuses a partial sequence of K39 protein, a partial sequence of K26 protein and the entire sequence of K9 protein; the recombinant protein is combined with a double antigen sandwich enzyme-linked immunosorbent assay and light-stimulated chemiluminescence technology to establish a clinical detection technology with high sensitivity, short time consumption and simple operation, thereby solving the technical problem that high sensitivity and high detection efficiency cannot be taken into account in the current commercial products for detecting canine leishmania antibodies.

[0022] The present invention designs and expresses a recombinant canine leishmaniasis diagnostic antigen with a size of 45KD. Compared with the recombinant protein obtained by the traditional single antigen protein, it has better immunogenicity. The colloidal gold detection card established by the present invention has a high analytical sensitivity, and can still significantly detect positive for the positive quality control serum after dilution 128 times; the detection card can be placed in a room temperature environment for 14 months and has good stability; the sensitivity of the detection card of the present invention is 86.4%, which is significantly higher than the commercial reference product. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the result of SDS-PAGE identification of the recombinant protein.

[0024] Figure 2 Schematic diagram of the lateral flow immunochromatographic detection process.

[0025] Figure 3 Schematic diagram of the detection of Leishmania antibodies in canine serum. DETAILED DESCRIPTION

[0026] The present invention is described in detail below by means of specific examples. The experimental methods in the following examples are conventional methods unless otherwise specified. The medicinal materials, reagent materials, etc. used in the following examples are commercially available products unless otherwise specified.

[0027] Table 1 is the amino acid sequence and nucleotide sequence table

[0028]

[0029]

[0030] The present invention is further described below by way of examples and drawings.

[0031] Example 1 Preparation of recombinant protein

[0032] A method for preparing a recombinant protein for detecting Leishmania comprises the following steps:

[0033] (1) Construction of recombinant protein expression vector

[0034] In order to construct a recombinant expression vector for diagnostic antigens, it was determined that the partial sequences of the three proteins K39, K26 and K9 were fused and expressed. The relevant amino acid sequences and nucleic acid sequences were retrieved from NCBI (National Center for Biotechnology Information), and the amino acid sequences were scored by linear epitope prediction (BepiPred 1.0 Server). Based on the prediction results and related existing studies, the antigen epitope sequences with high scores for each of the three proteins were selected as the fusion part, thereby determining:

[0035] The amino acid sequence of the recombinant protein is formed by fusion of a partial sequence of the K39 protein, a partial sequence of the K26 protein and the entire sequence of the K9 protein. The amino acid sequence of the recombinant protein is shown in SEQ ID NO.1;

[0036] The coding gene is recombined from a partial sequence of K39 nucleotides, a partial sequence of K26 nucleotides and a complete sequence of K9 nucleotides, and the nucleotide sequence of the coding gene is shown in SEQ ID NO.2;

[0037] We commissioned Global Gene Biotech to synthesize the nucleic acid sequence and introduce it into the pET-28a vector plasmid to connect the target gene of the diagnostic antigen to form a recombinant expression plasmid for the diagnostic antigen;

[0038] Table 1 Amino acid and nucleotide sequence list

[0039]

[0040]

[0041] (2) Inducible expression of recombinant protein

[0042] The recombinant expression plasmid pET-28a diagnostic antigen was transferred into the expression host competent cell E.coli BL21; a single positive bacterium was picked out on the plate and inoculated into LB liquid medium containing kanamycin sulfate, and activated overnight in a constant temperature shaking incubator at 25°C, and the bacterial solution was transferred to a new LB liquid medium containing kanamycin sulfate at a dilution of 1:100 (V / V), and cultured at 37°C and 250rpm for 2-3h until the logarithmic growth phase of E. coli, and IPTG with a concentration of 1mmol / L was added to induce protein expression, and cultured in a constant temperature shaking incubator at 20°C and 150rpm for 3-6h;

[0043] The induced expression bacterial solution was centrifuged at 5000g, 4°C, and 5min; after centrifugation, it was washed with PBS and the centrifugation operation was repeated; the wet bacteria were weighed and added at a ratio of 1g wet bacteria to 15mL lysis buffer, and 10mg lytic enzyme and 937.5U of universal nuclease (250U / μL) were added; after sufficient shaking to suspend the bacteria, the beaker was placed in a shaker at 4°C and 150rpm for lysis for 25min; the lysate was centrifuged at 16000g and 4°C for 25min, and the supernatant was purified;

[0044] (3) Purification of recombinant protein

[0045] In order to obtain a high-purity target protein, the supernatant obtained in the above steps is purified by nickel ion affinity chromatography. The nickel chloride in the nickel column can bind to the His tag on the fusion recombinant protein and also bind to imidazole. When the His-tag tag binds to the nickel ion medium in the bead hole on the surface of the chromatographic column, different concentrations of imidazole solution will pass through the chromatographic column to elute the impurity protein and fusion recombinant protein coordinated with nickel. Use 10 column volumes of binding buffer to balance His Bind and wash away the ethanol solution in the chromatographic column; slowly add the supernatant after lysis centrifugation, then elute the impurity protein with 5 column volumes of elution buffer, and finally elute the target recombinant protein with 10 column volumes of elution buffer, and collect them in 1.5mL centrifuge tubes in turn; after collection, use the nanodrop instrument to measure the protein concentration in the eluate and combine the eluate with a concentration of 0.2mg / mL and dialyze to obtain the recombinant protein.

[0046] Example 2 Identification of recombinant protein

[0047] The SDS polyacrylamide gel electrophoresis test is used to identify whether the molecular weight of the obtained recombinant protein meets the expectations. SDS combines with protein molecules to form a negatively charged protein-SDS complex. After the electrophoresis instrument is powered on, the protein-SDS complex moves to the electrode with uneven charge. According to the role of polyacrylamide gel molecular sieve, the migration rate of the complex is negatively correlated with the molecular weight of the protein. In order to identify the expression of the recombinant protein, the SDS-PAGE method is used to identify the purity of the protein. The specific steps are as follows:

[0048] (1) Adjusting the gel: Install the finished gel plate into the electrophoresis tank, slowly pull out the comb, and add electrophoresis buffer to cover the sample wells.

[0049] (2) Sample spotting: Pipette 10 μL of the dialyzed protein sample and spot it on the gel well. Make sure the tip of the pipette is inserted into the bottom layer of the well to prevent the sample from being added outside. Use a protein marker with a molecular weight of 50 KD.

[0050] (3) Electrophoresis: Connect the positive and negative electrodes of the electrophoresis instrument and turn on the power. Keep the voltage constant at 120V until the marker reaches the bottom of the gel plate.

[0051] (4) Staining: After the electrophoresis is completed, pry open the plastic sealing plate and place the gel in a rapid staining and destaining machine for 15 minutes. Remove the gel and observe the position of the marker corresponding to the gel band. The result is as follows: Figure 1 As shown by Figure 1 It can be seen that the molecular weight is around 45KD, which is consistent with the expected results, indicating that the experiment successfully expressed the recombinant protein after fusion.

[0052] Example 3 Assembly of colloidal gold test card

[0053] Test strip assembly Figure 2 As shown, it mainly consists of five parts: sample pad, gold label pad, NC membrane, water absorbent pad and PVC plate. The sample pad is Shanghai Liangxin SB08 glass cellulose membrane, which is soaked in a salt solution of 0.1MTris-HCl (pH8.5), 0.75% S9, 0.5% Tween-20, 1% BSA (MP), and 0.2% casein sodium, and then dried in a 45°C incubator overnight. The diagnostic antigen expressed in prokaryotes is marked at the NC membrane detection line (T line), and the goat anti-mouse monoclonal antibody is marked at the quality control line (C line).

[0054] During the test, add 20 μL of sample serum and three drops of sample dilution buffer to the assembled test card sample pad. After 10 minutes, observe the color development of the test line and quality control line with the naked eye.

[0055] Detection principle: The present invention uses a double antigen sandwich method to establish a lateral flow immunochromatography detection method for canine Leishmania antibodies. After the serum to be tested and the sample dilution buffer are dripped onto the test card, they are filtered for the first time at the sample pad to reach the gold label pad. The circulation of the liquid drives the colloidal gold particles labeled with the diagnostic antigen and the colloidal gold particles labeled with the non-interfering mouse monoclonal antibody on the gold label pad to continue moving forward, and in this process, the target antibody and the diagnostic antigen are combined to initially form a target antibody-diagnostic antigen-colloidal gold particle complex. When it reaches the detection line of the NC membrane, since the antibody has the characteristic of bivalent binding, the free end of the antibody in the above-mentioned complex will bind to the diagnostic antigen labeled on the detection line again, thereby forming a diagnostic antigen-target antibody-diagnostic antigen-colloidal gold particle complex attached to the detection line, thereby causing the detection line to develop color. The remaining components continue to move forward with the flow of the liquid. After reaching the quality control line, the labeled non-interfering mouse monoclonal antibody binds to the sheep anti-mouse secondary antibody labeled at the quality control line, thereby causing the quality control line to develop color.

[0056] Example 4 Detection of Leishmania antibodies

[0057] (1) Use a dropper to add 1 drop (about 20 μL) of the serum to be tested [centrifuged at 3000 rpm for 5 min at room temperature] into the sample well of the test card.

[0058] (2) Add 3 drops of sample diluent into the sample well.

[0059] (3) After reacting for 10 min at room temperature (25±5°C), observe the test results. Figure 3 shown.

[0060] Positive: C line is colored and T line is visible to the naked eye. It is considered positive regardless of the color depth.

[0061] Negative: C line shows color, T line does not show color, it is judged as negative;

[0062] Invalid: If the C line does not show color, it is considered invalid regardless of whether the T line shows color.

[0063] Example 5 Sensitivity and specificity

[0064] The sensitivity and specificity were evaluated by testing the results of different samples, and the results are shown in Table 2. Taking the test results of the commercial indirect ELISA kit produced by ID-Vet as the standard, for 72 positive samples, the present invention detected 61 cases, with a detection rate of 84.7%; the colloidal gold test card produced by InBios detected 55 cases, with a detection rate of 76.4%; for 30 negative samples, the present invention detected 1 positive case, and the colloidal gold test card produced by InBios detected 1 positive case, with specificity of 96.7%. It can be seen from the comparison that the present invention has higher sensitivity than the colloidal gold test card produced by InBios. It is also worth mentioning that although one case of both was detected as positive in the specificity evaluation, it was not the same sample. The present invention had an overlap with a case of Babesia trichomonas, and the colloidal gold test card of InBios had an overlap with a case of Salmonella.

[0065] Table 2 Comparison of the present invention and the reference method

[0066]

[0067] In view of the fact that false positives cannot be completely eliminated in canine Leishmania serological detection and the complicated experimental operation of ELISA detection, the present invention adopts a double antigen sandwich immunoassay with high specificity. The double antigen sandwich detection method is introduced into the lateral flow immunochromatography method, which can effectively reduce the occurrence of false positives in the detection from the method level. Compared with the single specific recognition mechanism of methods such as indirect ELISA and non-sandwich lateral flow immunochromatography, the double antigen sandwich can use the bivalent binding characteristics of antibodies for two specific recognitions, so it has the potential to improve specificity from a theoretical level. In addition, compared with indirect ELISA using a secondary antibody to detect only IgG, the double antigen sandwich method can effectively identify all antibody types, especially the recognition ability of IgM, which gives this method a unique advantage in the diagnosis of early cases of infection.

[0068] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of implementation of the present invention. If the present invention is modified or replaced by equivalents without departing from the spirit and scope of the present invention, it should be included in the protection scope of the claims of the present invention.

Claims

1. A recombinant protein for detecting Leishmania, characterized in that: The amino acid sequence of the recombinant protein is formed by fusion of a partial sequence of the K39 protein, a partial sequence of the K26 protein and the entire sequence of the K9 protein. The amino acid sequence of the recombinant protein is shown in SEQ ID NO.

1.

2. A gene encoding a recombinant protein of Leishmania, characterized in that: The coding gene is recombined from a partial sequence of K39 nucleotides, a partial sequence of K26 nucleotides and a complete sequence of K9 nucleotides, and the nucleotide sequence of the coding gene is shown in SEQ ID NO.

2.

3. The present invention provides an expression vector, characterized in that: Including the encoding gene.

4. An expression vector according to claim 3, characterized in that: The expression vector is pET-28a.

5. A host cell, characterized in that: Including the expression vector.

6. A host cell according to claim 5, characterized in that: The host cell is a eukaryotic host cell or a prokaryotic host cell.

7. A host cell according to claim 6, characterized in that: The host cell is a prokaryotic host cell.

8. A host cell according to claim 7, characterized in that: The host cell is preferably Escherichia coli.

9. A method for preparing a recombinant protein for detecting Leishmania, characterized in that: The method comprises the step of inducing the host cell to express protein.

10. Use of the recombinant protein for detecting Leishmania according to claim 1 in preparing a kit.

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