Canine Leishmania recombinant protein and its encoding gene, preparation method and application

Abstract

The present invention discloses a canine Leishmania recombinant protein and its encoding gene, preparation method and application, belonging to the field of genetic engineering. The canine Leishmania recombinant protein comprises an amino acid sequence shown in SEQ ID No.5 or SEQ ID No.7, and its encoding gene comprises a nucleotide sequence shown in SEQ ID No.6 and SEQ ID No.8, respectively. The present invention overcomes the shortcomings of the existing detection method, such as high equipment requirements, long detection time, and unsuitability for routine detection. The protein can be used for the preparation of colloidal gold detection reagent strips, and has the characteristics of high sensitivity, good specificity, strong stability, and simple operation, and is suitable as a conventional detection method for canine Leishmaniasis.

Description

Technical Field

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

[0002] Leishmaniasis is a zoonotic parasitic disease caused by Leishmania spp. amastigotes parasitizing mammalian macrophages. Leishmaniasis can be classified into three types, namely cutaneous, mucosal and visceral leishmaniasis, according to their different clinical manifestations. Visceral leishmaniasis (also known as kala-azar) can be fatal.

[0003] Visceral leishmaniasis can be divided into two types, namely human-derived and animal-derived. For the latter, dogs are the main source of infection and are the most widely distributed. Among the provinces (regions) currently prevalent in my country, four provinces are animal-derived, and dogs are the main source of infection. Leishmania donovani is the pathogen of visceral leishmaniasis. The management of dogs is the key to controlling this type of kala-azar. Therefore, it is of great significance to study effective, rapid, simple and field-friendly methods for detecting canine Leishmania infection to control this type of kala-azar.

[0004] Currently, the experimental diagnosis or detection of canine leishmaniasis mainly includes etiological methods, immunological methods and molecular biological methods.

[0005] Etiological diagnosis methods include microscopy, culture, animal inoculation, and histopathological examination. Microscopy is based on the detection of pathogens, so it has high specificity and is the gold standard method, but its detection rate depends on the level of worm load. The detection rate of asymptomatic infected dogs is much lower than that of symptomatic infected dogs, so its sensitivity is greatly limited. The culture method is to inoculate the puncture material into the culture medium and then examine it under the microscope after culture, which can improve the detection rate. However, the culture method is time-consuming and labor-intensive, requires strict aseptic operation, and has high technical requirements for operators. It is suitable for seed preservation and large-scale preparation of flagellates. The sensitivity of different tissue materials varies greatly. Animal inoculation is usually not used for diagnosis because it takes too long. Although histopathological examination has high specificity, it has low sensitivity and is not commonly used. Therefore, the etiological method has high specificity, but due to the wide distribution of Leishmania in dogs, the detection rate of single tissue sampling microscopy is often low, and the sampling and inspection process is relatively complicated, requiring high requirements for operators and low sensitivity.

[0006] The main immunological methods include direct agglutination test (DAT), indirect immunofluorescence antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA) and protein blotting. Although the direct agglutination test is economical and easy to operate, its specificity and sensitivity are not ideal. In addition, it is necessary to incubate and prepare multiple dilutions of serum, making it unsuitable for large-scale sample testing. The indirect immunofluorescence method can be used for epidemiological surveys, clinical diagnosis, and evaluation of post-treatment effects. However, it requires skilled operators and expensive equipment, and is not suitable for grassroots promotion and application. Enzyme-linked immunosorbent assay and protein blotting have high sensitivity and specificity, but their experimental conditions are demanding and the operation is complicated. They are only suitable for laboratory scientific research and cannot be used for grassroots promotion and application.

[0007] Molecular biological methods include polymerase chain reaction (PCR) and quantitative real-time PCR (qRT-PCR). Molecular biological methods have good specificity and sensitivity, but still have some limitations, such as the need for a thermal cycler and optimized experimental conditions, cumbersome product detection, and the need for professional personnel to operate, which is not conducive to on-site use.

[0008] Therefore, it is necessary to establish a simple, rapid and economical method to detect Leishmania. Summary of the invention

[0009] The present invention aims to overcome the shortcomings of existing detection methods, such as high equipment requirements, long detection time, and unsuitability for routine detection. A recombinant fusion protein A2-rkLO8 of canine Leishmania and its encoding gene, a preparation method and application are provided. The protein can be used for the preparation of colloidal gold detection reagent strips. The labeling technology has high sensitivity, good specificity, strong stability, and simple operation, and is suitable as a conventional detection method for canine Leishmania.

[0010] In order to achieve the above object, the present invention adopts the following technical solutions:

[0011] The first aspect of the present invention provides a canine Leishmania recombinant protein, comprising an amino acid sequence shown in SEQ ID No.5 or SEQ ID No.7.

[0012] In the present invention, the recombinant protein, fusion protein, recombinant fusion protein and fusion recombinant protein have the same meaning, and all refer to the expression product obtained by DNA recombination technology after two genes are recombined.

[0013] It can be seen that the recombinant protein is recombined from the rkLO8 protein including the amino acid sequence shown in SEQ ID No. 1 and the A2 protein fragment including the amino acid sequence shown in SEQ ID No. 3. The present invention fuses the full-length rkLO80 protein and the partial fragment of the A2 protein together for expression, which can not only improve the sensitivity of diagnosis, but also reduce cross-reactions with other pathogens.

[0014] In some embodiments of the present invention, the canine Leishmania recombinant protein consists of the amino acid sequence shown in SEQ ID No. 5 or SEQ ID No. 7. Preferably, the canine Leishmania recombinant protein consists of the amino acid sequence shown in SEQ ID No. 7, and has excellent sensitivity and specificity when detecting canine Leishmania.

[0015] The second aspect of the present invention provides a gene encoding a canine Leishmania recombinant protein, comprising a nucleotide sequence as shown in SEQ ID No.6 or SEQ ID No.8. The gene comprising the nucleotide sequence as shown in SEQ ID No.6 encodes a canine Leishmania recombinant protein comprising an amino acid sequence as shown in SEQ ID No.5; the gene comprising the nucleotide sequence as shown in SEQ ID No.8 encodes a canine Leishmania recombinant protein comprising an amino acid sequence as shown in SEQ ID No.7.

[0016] This gene sequence is to express the recombinant protein in E. coli, and the codons are optimized according to the preference of E. coli for codons. 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 gene sequence is codon optimized, which is suitable for E. coli expression and can improve the efficiency of protein expression.

[0017] The third aspect of the present invention provides an expression vector comprising the gene described in the second aspect of the present invention.

[0018] In some embodiments of the present invention, the expression vector is pET30a, which has kanamycin resistance, and the expressed fusion protein has a histidine (His) tag.

[0019] The fourth aspect of the present invention provides a host cell comprising the expression vector described in the third aspect of the present invention.

[0020] Furthermore, the host cell is a eukaryotic host cell or a prokaryotic host cell.

[0021] In some embodiments of the present invention, the host cell is a prokaryotic host cell. Preferably, the host cell is Escherichia coli, and more preferably, the Escherichia coli is BL21. Using Escherichia coli for expression has the advantages of short cycle, low cost, and high expression amount.

[0022] The fifth aspect of the present invention provides a method for preparing the canine Leishmania recombinant protein described in the first aspect of the present invention, characterized in that it includes the step of inducing the host cell described in the fourth aspect of the present invention to express the protein.

[0023] Furthermore, the host cell is a eukaryotic host cell or a prokaryotic host cell.

[0024] In some embodiments of the present invention, the host cell is a prokaryotic host cell. Preferably, the host cell is Escherichia coli, and more preferably, the Escherichia coli is BL21. Using Escherichia coli for expression has the advantages of short cycle, low cost, and high expression amount.

[0025] In some specific embodiments of the present invention, the step of inducing Escherichia coli to express protein is:

[0026] S1, culturing the Escherichia coli at 37° C. using LB medium containing 50 μg / mL kanamycin,

[0027] S2, when the OD600 of the E. coli culture reached 0.5-0.7, the expression was induced with IPTG at a final concentration of 1 mM. The induction conditions were: 28-32°C, 150-250 rpm, for 3-5 h;

[0028] S3, after induction, centrifuge and disrupt the cells to obtain the supernatant, purify, and renature the expressed canine Leishmania recombinant protein.

[0029] Preferably, in step S2, induction is performed when the OD600 of the E. coli culture solution reaches 0.6.

[0030] Preferably, in order to allow the recombinant protein to better maintain the active site, the present invention selects relatively mild culture and induction conditions, wherein the induction temperature during expression is 30°C, the induction speed is 200 rpm, and the induced IPTG concentration is 1 mM, so that the recombinant protein is expressed more slowly and has sufficient time for the formation of spatial conformation.

[0031] Preferably, in step S3, the bacteria are disrupted by ultrasonic disruption, with the conditions of 400w, 3s ultrasonication, and 6s interval. The disruption method of the present invention avoids the situation where the disruption is too severe and causes the loss of recombinant protein.

[0032] In the present invention, there are many ways to purify the recombinant protein, such as ion exchange chromatography, gel filtration chromatography, affinity chromatography, etc. In some embodiments of the present invention, affinity chromatography is selected, because a His tag is added to the recombinant protein, a one-step purification can achieve a higher purity.

[0033] Preferably, during purification, the loading buffer in the column buffer is 50mM Tris, 0.15M NaCl, pH 8.0, and the elution buffer is 50mM Tris, 0.15M NaCl, 0.5M Imidazole, pH 8.0; after purification, the collected target protein is dialyzed with 10mM PBS, pH 7.4, and the dialysate is changed every 12 hours for a total of 3 times. After dialysis, the protein solution is filtered and sterilized with a 0.22μm filter, and the protein concentration is determined by the Bradford method, and stored at -20°C for future use.

[0034] The sixth aspect of the present invention provides use of the canine Leishmania recombinant protein described in the first aspect of the present invention in the preparation of a kit for detecting canine Leishmania antibodies.

[0035] The seventh aspect of the present invention provides a kit for detecting canine Leishmania antibodies, characterized in that it comprises the canine Leishmania recombinant protein described in the first aspect of the present invention.

[0036] Furthermore, the kit also includes mouse IgG and goat anti-mouse IgG.

[0037] In some embodiments of the present invention, the double antigen sandwich gold label method is used to detect canine Leishmania antibodies.

[0038] Colloidal gold-labeled immunoassay is a new analytical technology that has emerged and developed rapidly in recent years. It is quick and simple, low-cost, pollution-free, and does not require training. Compared with traditional methods, it is more suitable for on-site testing. Compared with traditional methods, it has the advantages of short color development time and no need for expensive instruments, and has broad market prospects and application value.

[0039] In some specific embodiments of the present invention, the kit includes a double antigen sandwich gold label test strip, and the reagent method of the test strip is as follows:

[0040] (1) Add 1000 mL of ultrapure water to a conical flask, heat to boiling on a magnetic heating stirrer, then add 1 mL of 10% chloroauric acid, then add 1 mL of 10% trisodium citrate solution, continue heating and boiling for 15 min, and then cool to room temperature;

[0041] (2) Add 0.2 M K2CO3 to the cooled gold water to adjust the pH to 8.0, add goat anti-dog IgG H&L (Abcam, ab112849), stir for 15 min, then add BSA solution, stir again for 30 min, centrifuge at 10000 rpm for 20 min, and resuspend the precipitate with gold label diluent;

[0042] (3) Add 0.2 M K2CO3 to the gold water to adjust the pH to 7.0, add mouse IgG, stir for 15 min, add BSA solution, stir again for 30 min, centrifuge at 10000 rpm for 20 min, and resuspend the precipitate with gold label diluent;

[0043] (4) mixing the gold solutions labeled in steps 2 and 3, soaking the glass fiber, and freeze-drying to obtain a gold-labeled pad;

[0044] (5) Diluting canine Leishmania recombinant proteins rkLO8, A2, rkLO8-A2 and A2-rkLO8 to 0.5 mg / mL respectively with a spotting diluent, diluting goat anti-mouse IgG polyclonal antibody to 1.0 mg / mL with the same diluent, streaking the above two diluted solutions onto a nitrocellulose membrane, and drying at 37°C overnight. The spotting diluent is 10 mM PBS, 1% sucrose, pH 7.4;

[0045] (6) Assemble the above gold label pad, coated nitrocellulose membrane and filter paper, sample pad, card shell, etc. into a colloidal gold detection reagent strip.

[0046] When using, drop the canine biological sample onto the sample pad and place it at room temperature for 10 minutes before determining the test result. The determination criteria are as follows:

[0047] ① Only one band appears on the quality control line, and no band appears in the test area, which is negative;

[0048] ② Two bands appear, one in the quality control area and the other in the test area, which is positive;

[0049] ③ If no band appears on the quality control line, it indicates that the test strip is damaged and a new test strip should be replaced and retested.

[0050] In some embodiments of the present invention, the biological sample is serum or plasma, or any other body fluid that may contain antibodies.

[0051] Beneficial effects of the present invention

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] (1) The canine Leishmania recombinant protein of the present invention is obtained in an E. coli expression system, which has the advantages of short production cycle, high yield, low cost and good specificity;

[0054] (2) The His tag on the recombinant protein facilitates one-step purification to achieve a higher purity, and can exhibit better activity without enzyme cleavage;

[0055] (3) The recombinant fusion protein can be used as part of a colloidal gold detection kit for canine Leishmania, which has high sensitivity, good specificity, good stability, and is easy to operate, making it suitable as a routine detection method.

[0056] (4) The present invention overcomes the shortcomings of existing detection methods, such as high equipment requirements, long detection time, and unsuitability for routine detection. The protein can be used for the preparation of colloidal gold detection reagent strips, which have the characteristics of high sensitivity, good specificity, strong stability, and simple operation, and are suitable as a routine detection method for canine leishmaniasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 The results of the detection of the inducible expression of the recombinant proteins are shown: A: rkLO8; B: A2; C: rkLO8-A2 and A2-rkLO8.

[0058] Figure 2 The results of the indirect gold labeling method using four recombinant proteins to detect canine Leishmania antibodies are shown. DETAILED DESCRIPTION

[0059] Unless otherwise indicated, implied from the context, or customary in the prior art, all parts and percentages in this application are based on weight, and the tests and characterization methods used are all synchronized with the filing date of this application. Where applicable, the contents of any patent, patent application or disclosure involved in this application are fully incorporated herein by reference, and their equivalent patent families are also introduced as references, especially the definitions of relevant terms in the art disclosed in these documents. If the definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition of the term provided in this application shall prevail.

[0060] Numerical ranges in this application are approximate values, so unless otherwise specified, they may include numerical values ​​outside the range. Numerical ranges include all numerical values ​​from the lower limit to the upper limit increased by 1 unit, provided that there is an interval of at least 2 units between any lower value and any higher value. For a range comprising a numerical value less than 1 or a range comprising a fraction greater than 1 (e.g., 1.1, 1.5, etc.), 1 unit is appropriately considered as 0.0001, 0.001, 0.01 or 0.1. For a range comprising a single digit less than 10 (e.g., 1 to 5), 1 unit is generally considered as 0.1. These are only specific examples of what is intended to be expressed, and all possible combinations of numerical values ​​between the lowest and highest values ​​listed are considered to be clearly recorded in this application.

[0061] The terms "comprising", "including", "having" and their derivatives do not exclude the presence of any other components, steps or processes, and are irrelevant to whether these other components, steps or processes are disclosed in this application. To eliminate any doubt, unless explicitly stated, all compositions using the terms "comprising", "including", or "having" in this application may include any additional additives, adjuvants or compounds. On the contrary, except for those necessary for operating performance, the term "essentially consisting of..." excludes any other components, steps or processes from the scope of any description of the term below. The term "consisting of..." does not include any components, steps or processes that are not specifically described or listed. Unless explicitly stated, the term "or" refers to the listed individual members or any combination thereof.

[0062] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments.

[0063] Example

[0064] The following examples are used to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention and therefore can be considered as preferred embodiments of the present invention. However, it will be appreciated by those skilled in the art based on this specification that many modifications may be made to the specific embodiments disclosed herein and still achieve the same or similar results without departing from the spirit or scope of the present invention.

[0065] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the disclosure and materials cited therein are hereby incorporated by reference.

[0066] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many technical equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0067] The molecular biology experimental methods not specifically described in the following examples are all carried out in accordance with the specific methods listed in the book Molecular Cloning Experiment Guide (4th Edition) (J. Sambrook, MR Green, 2017), or in accordance with the kits and product instructions. Other experimental methods are conventional methods unless otherwise specified. The instruments and equipment used in the following examples are conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0068] Example 1 Construction of an expression vector containing a recombinant protein gene of Leishmania canis

[0069] In order to prepare a kit for detecting canine Leishmania with excellent sensitivity, specificity and stability, the inventors selected rkLO8 protein and A2 protein to construct a fusion protein.

[0070] The amino acid sequence of rkLO8 protein is as follows (SEQ ID No. 1):

[0071] ELATEWEDALRERALAERDEAAAAELDAAASTSQNARESASERLTSLEQLLRESEERAGELASQLESTTAAKMSAEQDRENTRATLEQQLRDSEERAAELASQLEATAAAKSSAEQDRENTRAALEQQLRDSEERAAELASQLESTT AAKTSAEQDRENTRATLEQQLRDSEERAAELASQLESTTAAKMSAEQDRENTRAALEQQLLESEERAGELASQLESTTAAKMSAEQDRENTRAALEQQLRDSEERAAELASQLESTTAAKMSAEQDRENTRATLEQQLRDSEERAAE

[0072] The gene sequence encoding rkLO8 protein was designed based on the protein sequence of NCBI Gene bank AGL98402.1, and its nucleotide sequence is as follows (SEQ ID No. 2):

[0073] GAACTGGCGACCGAATGGGAAGATGCGCTGCGCGAACGCGCGCTGGCGGAACGCGATGAAGCGGCGGCGGCGGAACTGGATGCGGCGGCGAGCACCAGCCAGAACGCGCGCGAAAGCGCGAGCGAACGCCTGACCAGCCTGGAACAGCTGCTGCGCGAAAGCGAAGAACGCGCGGGCGAACTGGCGAGCCAGCTGGAAAGCACCACCGCGGCGAAAATGAGCGCGGAACAGGATCGCGAAAACACCCGCGCGACCCTGGAACAGCAGCTGCGCGATAGCGAAGAACGCGCGGCGGAACTGGCGAGCCAGCTGGAAGCGACCGCGGCGGCGAAAAGCAGCGCGGAACAGGATCGCGAAAACACCCGCGCGGCGCTGGAACAGCAGCTGCGCGATAGCGAAGAACGCGCGGCGGAACTGGCGAGCCAGCTGGAAAGCACCACCGCGGCGAAAACCAGCGCGGAACAGGATCGCGAAAACACCCGCGCGACCCTGGAACAGCAGCTGCGCGATAGCGAAGAACGCGCGGCGGAACTGGCGAGCCAGCTGGAAAGCACCACCGCGGCGAAAATGAGCGCGGAACAGGATCGCGAAAACACCCGCGCGGCGCTGGAACAGCAGCTGCTGGAAAGCGAAGAACGCGCGGGCGAACTGGCGAGCCAGCTGGAAAGCACCACCGCGGCGAAAATGAGCGCGGAACAGGATCGCGAAAACACCCGCGCGGCGCTGGAACAGCAGCTGCGCGATAGCGAAGAACGCGCGGCGGAACTGGCGAGCCAGCTGGAAAGCACCACCGCGGCGAAAATGAGCGCGGAACAGGATCGCGAAAACACCCGCGCGACCCTGGAACAGCAGCTGCGCGATAGCGAAGAACGCGCGGCGGAA

[0074] The amino acid sequence of the A2 protein is as follows (SEQ ID No. 3):

[0075] MKIRSVRPLVVLLVCVAAVLALSASAEPHKAAVDVGPLSVDVGPLSVGPQSVGPLSVGPQSVGPLSVDVGPLSVGPQSVGPLSVDVGPLSVGPQSVGPLSVGPQAVGPLSVGPQSVGPLSVDVGPQAVGPQSVGPLSVGPQSVGPLSVGPQSVGPLSVGPLSVGPQSVGSLSVGPQSVGPLSVGP

[0076] The above sequence is obtained by analyzing the antigenic epitopes of the full length of the A2 protein and selecting the most dominant antigenic epitope. The nucleotide sequence of the gene encoding the A2 protein is as follows (SEQ ID No. 4):

[0077] ATGAAAATTCGCAGCGTGCGCCCGCTGGTGGTGCTGCTGGTGTGCGTGGCGGCGGTGCTGGCGCTGAGCGCGAGCGCGGAACCGCATAAAGCGGCGGTGGATGTGGGCCCGCTGAGCGTGGATGTGGGCCCGCTGAGC GTGGGCCCGCAGAGCGTGGGCCCGCTGAGCGTGGGCCCGCAGAGCGTGGGCCCGCTGAGCGTGGATGTGGGCCCGCTGAGCGTGGGCCCGCAGAGCGTGGGCCCGCTGAGCGTGGATGTGGGCCCGCTGAGCGTGGGCC CGCAGAGCGTGGGCCCGCTGAGCGTGGGCCCGCAGGCGGTGGGCCCGCTGAGCGTGGGCCCGCAGAGCGTGGGCCCGCTGAGCGTGGATGTGGGCCCGCAGGCGGTGGGCCCGCAGAGCGTGGGCCCGCTGAGCGTGGG CCCGCAGAGCGTGGGCCCGCTGAGCGTGGGCCCGCAGAGCGTGGGCCCGCTGAGCGTGGGCCCGCTGAGCGTGGGCCCGCAGAGCGTGGGCAGCCTGAGCGTGGGCCCGCAGAGCGTGGGCCCGCTGAGCGTGGGCCCG

[0078] In order to further improve the sensitivity and specificity of detection, the inventors fused the above two proteins. There are two fusion methods: one is to place rkLO8 at the N-terminus and A2 at the C-terminus, that is, rkLO8-A2 fusion protein; the other is to place A2 at the N-terminus and rkLO8 at the C-terminus, that is, A2-rkLO8 fusion protein.

[0079] The amino acid sequence of the rkLO8-A2 fusion protein is as follows (SEQ ID No.5):

[0080] ELATEWEDALRERALAERDEAAAAELDAAASTSQNARESASERLTSLEQLLRESEERAGELASQLESTTAAKMSAEQDRENTRATLEQQLRDSEERAAELASQLEATAAAKSSAEQDRENTRAALEQQLRDSEERAAELASQLESTTAAKTSAEQDRENTRATLEQQLRDSEERAAELASQLESTTAAKMSAEQDRENTRAALEQQLLESEERAGELASQLESTTAAKMSAEQDRENTRAALEQQLRDSEERAAELASQLESTTAAKMSAEQDRENTRATLEQQLRDSEERAAEMKIRSVRPLVVLLVCVAAVLALSASAEPHKAAVDVGPLSVDVGPLSVGPQSVGPLSVGPQSVGPLSVDVGPLSVGPQSVGPLSVDVGPLSVGPQSVGPLSVGPQAVGPLSVGPQSVGPLSVDVGPQAVGPQSVGPLSVGPQSVGPLSVGPQSVGPLSVGPLSVGPQSVGSLSVGPQSVGPLSVGP

[0081] The nucleotide sequence of the gene encoding the rkLO8-A2 fusion protein is as follows (SEQ ID No.6):

[0082]

[0083] The amino acid sequence of the A2-rkLO8 fusion protein is as follows (SEQ ID No.7):

[0084] MKIRSVRPLVVLLVCVAAVLALSASAEPHKAAVDVGPLSVDVGPLSVGPQSVGPLSVGPQSVGPLSVDVGPLSVGPQSVGPLSVDVGPLSVGPQSVGPLSVDVGPLSVGPQAVGPLSVGPQSVGPLSVDVGPQAVGPQSVGPLSVGPQSVGPLSVGPQSVGPLSVGPLSVGPQSVGSLSVGPQSVGPLSVGPELATEWEDALRERALAERDEAAAAELDAAASTSQNARESASERLTSLEQLLRESEERAGELASQLESTTAAKMSAEQDRENTRATLEQQLRDSEERAAELASQLEATAAAKSSAEQDRENTRAALEQQLRDSEERAAELASQLESTTAAKTSAEQDRENTRATLEQQLRDSEERAAELASQLESTTAAKMSAEQDRENTRAALEQQLLESEERAGELASQLESTTAAKMSAEQDRENTRAALEQQLRDSEERAAELASQLESTTAAKMSAEQDRENTRATLEQQLRDSEERAAE

[0085] The nucleotide sequence of the gene encoding the A2-rkLO8 fusion protein is as follows (SEQ ID No.8):

[0086]

[0087] The gene sequence of the above recombinant fusion protein has been codon-optimized and synthesized by General Biosystems (Anhui) Co., Ltd. The synthesized gene was inserted into the E. coli expression vector pET30a(+) by genetic engineering to form a recombinant expression vector, which is kanamycin-resistant and fused with a histidine tag.

[0088] Example 2 Expression of four canine Leishmania recombinant proteins

[0089] (1) The four recombinant expression vectors in Example 1 were transformed into Escherichia coli BL21 cells to obtain recombinant strains;

[0090] (2) The recombinant strain was cultured in LB medium at 37°C in a shaking flask until the bacterial concentration reached OD = 0.5-0.7, and 1 mM IPTG was added to induce expression at 30°C and 200 rpm for 3-5 hours;

[0091] (3) After induction, centrifuge and disrupt the cells to obtain the supernatant and purify the expressed canine Leishmania recombinant protein.

[0092] The E. coli BL21 expression system has the characteristics of short cycle, low cost and large expression amount. In order to make the recombinant protein better maintain the active site, the inventors selected relatively mild culture and induction conditions. The induction temperature during expression was 30°C, the induction speed was 200rpm, and the induced IPTG concentration was 1mM. This made the recombinant protein express more slowly and have sufficient time to form a spatial conformation.

[0093] Figure 1 The results of SDS-PAGE gel electrophoresis of bacterial proteins induced by IPTG for recombinant expression showed that there was no significant difference in the protein band of the expected size position (30 kDa) of the rkLO8 recombinant protein before and after induction. Figure 1 A), there is no significant difference in the protein band at the expected size position (18 kDa) of the A2 recombinant protein ( Figure 1 In middle B), there is no significant difference in the protein bands at the expected size position of the recombinant proteins rkLO8-A2 (50 kDa) and A2-rkLO8 (50 kDa) ( Figure 1 Middle C).

[0094] Example 3 Purification and refolding of recombinant protein containing canine Leishmania

[0095] The bacteria were broken by ultrasonic disruption, with the conditions of 400w, 3s ultrasonic, 6s interval, 50mM Tris, 0.15M NaCl, pH 8.0 in the column buffer, and 50mM Tris, 0.15M NaCl, 0.5MImidazole, pH 8.0 in the elution buffer; the target protein collected after purification was dialyzed with 10mM PBS, pH 7.4, and the dialysate was changed every 12h, for a total of 3 times, and the protein solution after dialysis was filtered and sterilized with a 0.22μm filter, and the protein concentration was determined by the Bradford method, and stored at -20°C for future use. Since the His tag was added to the above recombinant protein, one-step purification can achieve a higher purity, and no enzymatic removal of the tag is required, which reduces the steps and reduces the cost.

[0096] Example 4 Indirect gold labeling method for detecting canine Leishmania antibodies

[0097] 1. Preparation of colloidal gold test strips for canine Leishmania

[0098] (1) Add 1000 mL of ultrapure water to a conical flask and heat to boiling on a magnetic heating stirrer. Then add 1 mL of 10% chloroauric acid (sigma) and 1 mL of 10% trisodium citrate solution. Continue heating and boiling for 15 min, and then cool to room temperature.

[0099] (2) Add 0.2 M K2CO3 to the cooled gold water to adjust the pH to 8.0, add goat anti-dog IgG H&L (Abcam, ab112849), stir for 15 min, then add BSA solution, stir again for 30 min, centrifuge at 10,000 rpm for 20 min, and resuspend the precipitate in gold label diluent.

[0100] (3) Add 0.2 M K2CO3 to the gold water to adjust the pH to 7.0, add mouse IgG, stir for 15 min, add BSA solution, stir again for 30 min, centrifuge at 10000 rpm for 20 min, and resuspend the precipitate with gold label diluent;

[0101] (4) mixing the gold solutions labeled in steps 2 and 3, soaking the glass fiber, and freeze-drying to obtain a gold-labeled pad;

[0102] (5) Diluting canine Leishmania recombinant proteins rkLO8, A2, rkLO8-A2 and A2-rkLO8 to 0.5 mg / mL respectively with a spotting diluent, diluting goat anti-mouse IgG polyclonal antibody to 1.0 mg / mL with the same diluent, streaking the above two diluted solutions onto a nitrocellulose membrane, and drying at 37°C overnight. The spotting diluent is 10 mM PBS, 1% sucrose, pH 7.4;

[0103] (6) Assemble the above gold label pad, coated nitrocellulose membrane and filter paper, sample pad, card shell, etc. into a colloidal gold detection reagent strip.

[0104] The colloidal gold detection reagent strip prepared by the present invention is an indirect method colloidal gold detection reagent strip. Compared with other detection methods, it has the advantages of being convenient and fast, specifically sensitive, highly stable, not requiring special equipment and reagents, and having intuitive result judgment, and is suitable as a conventional detection method.

[0105] The gold label diluent is 20 mM Tris, 0.5% BSA, 0.1% TritonX-100, 0.01% Proclin300, pH 8.0 solution.

[0106] 2. Detection of canine Leishmania colloidal gold test strips

[0107] Add 10 μL of the sample to be tested (serum, plasma) to the sample pad, then add the sample diluent, and let it stand at room temperature for 10 minutes before determining the result. The result determination criteria are as follows:

[0108] ① Only one band appears on the quality control line, and no band appears in the test area, which is negative;

[0109] ② Two bands appear, one in the quality control area and the other in the test area, which is positive;

[0110] ③ If no band appears on the quality control line, it indicates that the test strip is damaged and a new test strip should be replaced and retested.

[0111] The canine Leishmania recombinant proteins rkLO8, A2, rkLO8-A2 and A2-rkLO8 were spotted on the membrane and assembled into colloidal gold test strips to test positive and negative sera. The test results were as follows: Figure 2 As shown in the figure, it can be clearly seen that the four test strips of positive and negative specimens are all in line with expectations, but the positive results show four different strengths, among which the result obtained by the rkLO8 dot membrane is the weakest, and the result obtained by the A2-rkLO8 fusion protein dot membrane is the strongest, which is significantly stronger than the result obtained by the rkLO8-A2 fusion protein dot membrane.

[0112] Example 5 Clinical specimen validation of four canine Leishmania colloidal gold detection reagent strips

[0113] In order to verify the sensitivity, specificity and accuracy of the four test cards, the inventors conducted a large number of specimens for verification. A total of 100 positive canine Leishmaniasis sera and 200 unaffected dog sera were tested. The test results of the four test strips are shown in Table 1, where the sensitivity is: true positive / (true positive + false negative) × 100%, the specificity is: true negative / (true negative + false positive) × 100%, and the accuracy is: (positive + negative) / total number of specimens × 100%.

[0114] Table 1 Clinical specimen verification of four colloidal gold test strips

[0115] Recombinant protein True Positive False Negative True Negative False Positive Sensitivity Specificity Accuracy rkLO8 74 26 180 20 74％ 90％ 84.7％ A2 86 14 184 16 86％ 92％ 90％ rkLO8-A2 90 10 186 14 90％ 93％ 92％ A2-rkLO8 95 5 197 3 95％ 98.5％ 97.3％

[0116] The data in Table 1 show that the four canine Leishmania recombinant proteins of the present invention have good sensitivity and specificity, among which the colloidal gold test strip made of the recombinant protein A2-rkLO8 dot film has the highest accuracy, indicating that the A2-rkLO8 recombinant fusion protein is very suitable as a raw material for making canine Leishmania antibody detection test strips; and the produced colloidal gold test strip has the advantages of high sensitivity and good specificity, short detection time, good stability, and simple operation. It only needs to be placed at room temperature for 10 minutes to determine the result, which is very suitable for use as a conventional detection method.

[0117] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A canine Leishmania recombinant protein, characterized in that: It consists of the amino acid sequence shown in SEQ ID No. 5 or SEQ ID No.

7.

2. A gene encoding a recombinant protein of canine Leishmania, characterized in that: It consists of the nucleotide sequence shown in SEQ ID No. 6 or SEQ ID No.

8.

3. An expression vector, characterized in that: Comprising the gene according to claim 2.

4. A host cell, characterized in that Contains the expression vector according to claim 3.

5. A method for preparing the canine Leishmania recombinant protein according to claim 1, characterized in that: The method comprises the step of inducing the host cell according to claim 4 to express protein.

6. The method according to claim 5, characterized in that The expression vector is pET30a, and the host cell is Escherichia coli.

7. The method according to claim 6, characterized in that The steps for inducing host cells to express proteins are: S1, culturing the Escherichia coli at 37° C. using LB medium containing 50 μg / mL kanamycin, S2, when the OD600 of the E. coli culture reached 0.5-0.7, the expression was induced with IPTG at a final concentration of 1 mM. The induction conditions were: 28-32°C, 150-200 rpm, for 3-5 h; S3, after induction, centrifuge and disrupt the cells to obtain the supernatant, purify, and renature the expressed canine Leishmania recombinant protein.

8. Use of the canine Leishmania recombinant protein according to claim 1 in the preparation of a kit for detecting canine Leishmania antibodies.

9. A kit for detecting canine Leishmania antibodies, characterized in that: The invention comprises the canine Leishmania recombinant protein as claimed in claim 1.

Images