CN110133290A - A kind of ELISA kit for diagnosing heartworm disease - Google Patents

Abstract

The invention relates to the field of biotechnology and discloses an ELISA kit for diagnosing heartworm disease. The kit of the invention comprises a solid-phase carrier coated with the small heat shock protein of Dirofilaria imimaginae. The present invention provides the related application of Dirofilaria imimaginum small heat shock protein as the antigen for diagnosis of heartworm disease. Relevant experimental results show that Dirofilaria imimaginum small heat shock protein can be recognized by serum of dogs naturally infected with heartworm disease , rabbit anti-IgG prepared from Dirofilaria imimaginum small heat shock protein can recognize Dirofilaria imimaginum small heat shock protein and has good immunogenicity and reactogenicity; meanwhile, it shows extremely high sensitivity and specificity in the indirect ELISA method Various results prove that the small heat shock protein of Dirofilaria imimaginum can be used as a diagnostic antigen for Dirofilariasis and can be applied to related detection kits.

Description

A kind of ELISA kit for diagnosing heartworm disease

technical field

The invention relates to the field of biotechnology, in particular to an ELISA kit for diagnosing heartworm disease.

Background technique

Dirofilariasis is a zoonotic parasitic disease transmitted by mosquitoes. Dirofilaria immitis mainly parasitizes the pulmonary artery and right ventricle of dogs and cats, and adult Dirofilaria immitis parasitic on the pulmonary artery can cause vascular disease and pulmonary hypertension in the host. Case reports of human heartworm infection have been on the rise in recent years due to increased mosquito vector mobility due to global warming.

The routine diagnostic methods for heartworm disease mainly include pathogen detection, serological detection and molecular biological detection. At present, serological detection is the most commonly used detection method, which is divided into antigen detection and antibody detection. False-negative results of antigen detection are common in cases of mild infection, immature females, and only infected males; the advantage of antibody detection over antigen detection is that both females and males can be detected. Two months after the host is infected with Dirofilaria imimaginae, the immune response produced by the larvae stimulating the body can be detected. Antibody detection can detect Dirofilaria imimaginum earlier than antigen detection, which is suitable for the early stage of heartworm disease detection. Exploring and discovering effective new diagnostic methods for heartworm disease has important practical significance for the efficient diagnosis of heartworm disease.

Contents of the invention

In view of this, the object of the present invention is to provide an ELISA kit for diagnosing heartworm disease, so that the kit has higher specificity and sensitivity in detection.

In order to realize the foregoing invention object, the present invention provides following technical scheme:

An ELISA kit for diagnosing heartworm disease comprises a solid phase carrier coated with heartworm small heat shock protein (Di-sHSP12.6). In a specific embodiment of the present invention, the solid-phase carrier can be selected as a 96-well culture plate or a solid-phase carrier similar to it, and the coating concentration of the heartworm small heat shock protein is 100 μl/well. The carrier is coated with the coating solution, and the coating solution is obtained by adjusting the pH to 9.6 with 0.39g Na ₂ CO ₃ , 35mM NaHCO ₃ , and 0.2M NaCl.

After determining the core components of the kit, the ELISA kit also includes one or more of enzyme-labeled secondary antibody, washing solution, color developing solution, blocking solution, diluent and stop solution.

Wherein, the enzyme-labeled secondary antibody is preferably HRP-labeled rabbit anti-dog IgG. In a specific embodiment of the present invention, the enzyme-labeled secondary antibody is a product purchased from Wuhan Boster Bioengineering Co., Ltd., and the enzyme-labeled secondary antibody The dilution ratio is 1:2000;

The washing solution is preferably PBS-T washing solution. In the specific embodiment of the present invention, the composition of the PBS-T washing solution is: 8gNaCl, 0.2gKCl, 1.42gNa ₂ HPO ₄ , 0.27gKH ₂ PO ₄ , dissolved in 800mL Add 0.5mL Tween20 to deionized water, and dissolve to 1L; the color developing solution is preferably TMB developing solution;

The blocking solution is preferably skim milk, and in a specific embodiment of the present invention, the concentration of the skim milk is 5%.

The stop solution is preferably sulfuric acid solution, the concentration is preferably 2mol/L; the preparation method is to slowly add 21.7mL of 98% concentrated sulfuric acid in 178mL of deionized water, cool to room temperature, and store at 4°C;

The diluent is preferably PBS; the preparation method is 8gNaCl, 0.2gKCl, 1.42gNa ₂ HPO ₄ , 0.27gKH ₂ PO ₄ , dissolved in 800mL deionized water, fixed to 1L, sterilized, and stored at room temperature.

Herein, the Dirofilaria imimaginum small heat shock protein may be non-natural, for example, synthesized or expressed by an artificial vector (commonly referred to as recombinant protein rDi-sHSP12.6 in the industry). The term "non-natural" means that the target substance is not naturally occurring in nature, which does not exclude that the non-natural substance has the same structure and/or composition as the naturally occurring substance.

In C. elegans, 14 small heat shock proteins (sHSPs) have been discovered, which can be divided into 6 groups according to the molecular mass: sHSP12s (including HSP12.1, HSP12.2, HSP12.3 and HSP12. 6), sHSP16s (HSP16.1, HSP16.2, HSP16.41, HSP16.48 and newly discovered F08H9.3, F08H9.4), HSP25, HSP43, HSP17.5 and stress-induced protein-1 (stress-induced protein-1, SIP-1), studies have confirmed that it can not only maintain the necessary protein spatial conformation of cells under stress conditions, protect cell life activities to maintain cell survival, but also play a role in protein folding, transmembrane transport, transport, body immunity, It plays an important role in basic functions such as apoptosis, cytoskeleton and nuclear skeleton stability. At present, there is no report on the related research of Dirofilaria imimaginum small heat shock protein (Di-sHSP12.6) in heartworm disease.

The present invention obtains the recombinant heartworm small heat shock protein (rDi-sHSP12.6, which has the same amino acid sequence as Di-sHSP12.6, as shown in SEQ ID NO: 1) through prokaryotic expression, and immunizes it Western blot, ELISA and establishment of early diagnostic methods. Western blot results showed that the rabbit anti-IgG prepared from recombinant protein rDi-sHSP12.6 could recognize the recombinant protein rDi-sHSP12.6, and the recombinant protein rDi-sHSP12.6 could be recognized by the serum of dogs naturally infected with heartworm disease, but not by healthy The recognition of canine serum shows that the small heat shock protein of Dirofilaria imimaginum has good immunogenicity and reactogenicity.

The results of indirect ELISA showed that 24 positive sera and 24 negative sera were detected by rDi-sHSP12.6 protein, and the results showed that 22 positive sera had OD450 > critical value, 2 positive sera and 24 positive sera Negative serum OD450<critical value (0.699), then the sensitivity of the method is estimated to be 91.6% (22/24); at the same time, it is not associated with canine Echinococcus granulosus positive serum, canine cysticercosis positive serum and canine hookworm positive serum. Serum (totally 18) cross-reaction occurs, only 2 of the 18 Toxocara canis positive sera detected have slight cross-reaction, then the specificity of the method is estimated to be 91.6% (34/36); in summary As mentioned above, rDi-sHSP12.6 has high sensitivity and specificity, low cross-reactivity rate, and good diagnostic effect. It is suitable as a diagnostic antigen for heartworm disease and as a diagnostic antigen for the preparation of related detection kits .

From the above technical solutions, it can be known that the present invention provides the relevant application of Dirofilaria imimaginum small heat shock protein as a diagnostic antigen for Dirofilaria immitis. The relevant experimental results show that Dirofilaria imimaginum small heat shock protein can be naturally infected by Dirofilaria imimaginae Canine serum recognition of canine worm disease, rabbit anti-IgG prepared by Dirofilaria imimaginum small heat shock protein can recognize Dirofilaria imimaginum small heat shock protein, and has good immunogenicity and reactogenicity; at the same time, it shows in the indirect ELISA method With extremely high sensitivity and specificity, various results prove that the small heat shock protein of Dirofilaria imimaginum can be used as a diagnostic antigen for Dirofilariasis and can be applied to related detection kits.

Description of drawings

Figure 1 shows the PCR amplification of Di-sHSP12.6 gene; wherein, M is DNA molecular quality standard (DL2000); 1-4: Di-sHSP12.6 PCR product;

Figure 2 shows the double-digestion identification of the recombinant plasmid pET32a(+)-sHSP12.6; among them, M: DNA molecular quality standard (DL2000); 1-4: double-digestion products of pET32a(+)-sHSP12.6 ;

Figure 3 shows the expression of recombinant Di-sHSP12.6 protein; among them, M: protein molecular mass standard; 1: E. coli induced by IPTG without pET32a(+)-sHSP12.6; 2-4: induced by IPTG Escherichia coli containing pET32a(+)-sHSP12.6;

Figure 4 shows the solubility analysis of rDi-sHSP12.6; among them, M: protein molecular mass standard; 1: 8M urea dissolved; 2: 4M urea dissolved; 3: 2M urea dissolved; 4: supernatant;

Figure 5 shows the expression, purification and western blot analysis of rDi-sHSP12.6; wherein, 1: IPTG-induced Escherichia coli that does not contain recombinant pET32a(+)-sHSP12.6; 2: IPTG-induced Escherichia coli containing recombinant pET32a( +)-sHSP12.6 Escherichia coli; 3: Rabbit anti-rDi-sHSP12.6 IgG purified by nickel column; 4: rDi-sHSP12.6 purified by nickel column; 5: natural infection with Dirofilaria imimaginae The canine positive serum specifically recognizes rDi-sHSP12.6; 6: The rabbit anti-rDi-sHSP12.6 IgG purified by nickel column recognizes rDi-sHSP12.6; 7: The healthy negative canine serum recognizes rDi-sHSP12.6 ;

Figure 6 shows the indirect immunofluorescence localization of Di-sHSP12.6 protein in cross-sections of female and male Dirofilaria immigatus; where, I: intestine; UT: uterus; TE: testis; MU: muscle; HY: lateral cord; PS : Pseudocoelom; Scale bar: 200 μm; A, B: female; C, D: male; Note: The green fluorescent area is the approximate position of protein distribution;

Figure 7 shows the indirect ELISA (specificity) of the recombinant protein rDi-sHSP12.6;

Figure 8 shows the indirect ELISA (sensitivity) of recombinant protein rDi-sHSP12.6.

Detailed ways

The invention discloses an ELISA kit for diagnosing heartworm disease. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters to realize it. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The kit of the present invention has been described through the examples, and relevant personnel can obviously make changes or appropriate changes and combinations to the kit described herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention .

The invention extracts the total RNA of the adult body of Dirofilaria imimaginae and reverse transcribes it into cDNA, and amplifies the encoding sequence of Dirofilaria imimaginum small heat shock protein (Di-sHSP12.6) from the cDNA. After T cloning, the amplified product was introduced into the expression vector by enzyme-cut ligation, and the recombinant rDi-sHSP12.6 was obtained by prokaryotic expression in Escherichia coli.

In the experiments of the specific implementation, all experimental animals were handled in strict accordance with the "Animal Protection Law of the People's Republic of China" (draft issued on September 18, 2009). All procedures were implemented in strict accordance with the "Guidelines for the Care of Experimental Animals of the Animal Ethics Committee of Sichuan Agricultural University" (Ya'an, China; approval number: 2013-028). All methods were performed in accordance with relevant guidelines and regulations, including any relevant details.

Dirofilaria immigatus parasites came from dogs that were naturally infected and autopsyed in Sichuan Province. The parasites were separated from the heart of the dog and washed with PBS. Referring to relevant literature, it was identified as Dirofilaria imimaginum by morphology. Dirofilariasis positive sera were collected from positive dogs naturally infected with Dirofilaria imimaginum in Sichuan Province. The serum used for cross-reaction was collected from dogs that had undergone fecal examination or autopsy after natural death (blood was collected before death); negative serum was collected from 3-month-old Beagle dogs (experimental dogs) that had been treated with internal and external deworming in spring. ).

The experimental animals were 2 healthy New Zealand rabbits, female, about 3 months old, weighing 1.5-2.0 kg, purchased from Chengdu Dashuo Biological Co., Ltd.

Strains and plasmids: Escherichia coli DH5α, Escherichia coli BL21(DE3), purchased from Tiangen Company; TaKaRa pMD19-TVector, purchased from Dalian Bao Biological Engineering Co., Ltd.; prokaryotic expression vector pET32a(+), purchased from Invitrogen.

An ELISA kit for diagnosing heartworm disease provided by the present invention will be further described below.

Example 1: Gene Amplification

1. Extraction of total RNA from Dirofilaria imimaginae

Prepare a pre-cooled mortar at -80°C in advance, take out the adult body of Dirofilaria imimilis (about 20 mg) from the liquid nitrogen, cut it into pieces, add a little liquid nitrogen and grind it thoroughly, and then follow the method of RNA extraction reagent from Tiangen animal tissue According to the instructions in the box, total RNA of Dirofilaria imimagina was extracted and stored at -80°C.

2. Synthesis of the first strand of cDNA

The first strand of cDNA was synthesized using the extracted total RNA of Dirofilaria imimaginae as a template and OligodT(18) as a primer, according to the instructions of the reverse transcription kit from Thermo Company.

3. Design and synthesis of primers

Referring to the nucleotide sequence of Filaria malayi sHSP12.6 in NCBI (gb:XM_001900555.1), primers were designed using Primerpremier5.0, and the underlined part is the enzyme cutting site (BamHI and XhoI):

Di-sHSP12.6 upstream primer (F): CGGATCCATGGAAGAAAAGGTAGTGGA

Di-sHSP12.6 downstream primer (R): CGAGCTCTCATGCTTTCTTTTTGGC

4. Amplification procedure and system

PCR amplification was carried out using Dirofilaria immigatus cDNA as substrate template. The amplification program is shown in Table 1, and the amplification system is shown in Table 2:

Table 1

Table 2

5. Product recovery

After the above products are subjected to nucleic acid gel electrophoresis, compare whether the band size is correct under the gel imaging system. Then cut out the target band under ultraviolet light, and perform DNA recovery on the target band according to the gel recovery kit of Tiangen Biotechnology Co., Ltd., and store at -20°C.

6. Amplification and basic physical and chemical properties of Di-sHSP12.6 gene

A band of about 340bp was amplified using Dirofilaria imimagina cDNA as a template (Figure 1, lanes 1-4), and the sequencing results of Sangon Biotech Co., Ltd. showed that the sequence had 100% homology with the sequence in NCBI.

The length of the Di-sHSP12.6 gene amplified by the present invention is 342bp. After amino acid sequence analysis, it encodes 113 amino acids (the last 3 bases are stop codons and does not encode amino acids), and its molecular mass is 13052.71. It is speculated that its molecular formula is C ₅₇₅ H ₉₁₀ N ₁₆₂ O ₁₇₉ S ₃ , ProtParam predicted that the Di-sHSP12.6 protein mainly contained Val, Thr, Lys and Asp. The signal peptide analysis results showed that Di-sHSP12.6 had no signal peptide, and the prediction of the transmembrane region showed that Di-sHSP12.6 was located in the extracellular region.

Embodiment 2: cloning and identification

1. Connection

Ligate the recovered DNA fragment of the target band with the pMD19-T vector, overnight at 16°C (no more than 16h), see Table 3 for the ligation system:

table 3

2. Conversion

Take out 8 μL of the ligated product and place it in a 1 mL EP tube, add 30 μL E.coliDH5α competent cells, mix gently with a micro-injector, ice-bath for 30 minutes, and then bathe in 42°C water for 90 seconds, then ice-bath for 5 minutes. Add 600μL LB liquid medium to the EP tube, incubate at 37℃, 180r/min for 1.5h, take 100μL of the bacterial solution and evenly inoculate it on the solid medium containing ampicillin (AMP) resistance, and place it in a constant temperature incubator at 37℃ Cultivate for 12h.

3. Sequencing identification

Select a single colony with normal morphology and disperse and inoculate it in 1 mL of LB liquid medium (containing 1 μL AMP), and culture it in an environment of 180 r/min at 37°C for 6 hours, and determine whether a band of the size of the target fragment can be amplified by PCR of the bacterial liquid, and will be able to amplify Take 400 μL of the bacterial liquid that produced the target fragment and send it to Sangon Biotech Co., Ltd. for sequencing. The bacterial solution PCR reaction system is shown in Table 4:

Table 4

Example 3: Construction of expression vectors

1. Extract plasmid

The bacterial liquid sequenced correctly in Example 2 was expanded and cultivated, and the Di-sHSP12.6 plasmid was extracted according to the instructions of the Tiangen plasmid mini-extraction kit, and finally 5 μL was mixed with 5 μL loading buffer for accounting gel electrophoresis to test the purity of the plasmid.

2. Enzyme digestion and recovery of target gene plasmid and expression vector plasmid

Digest the target gene plasmid and the expression vector pET32a(+) plasmid obtained according to the method in 2.3.1 with the same Takara fast cutting enzyme to obtain the same cohesive ends. Di-sHSP12.6 and pET32a(+) expression vector plasmids were digested with (BamHI and SacⅠ) fast cutting enzymes. According to the method in Example 1, gel recovery was performed on the DNA fragments of the target gene plasmid and the expression vector plasmid after enzyme digestion.

3. The target gene is connected to the expression vector

The DNA fragments recovered in 2 were respectively taken and ligated with T4 ligase to construct the pET32a(+)-recombinant protein expression vector plasmid. After brief centrifugation, connect in a PCR machine at 22°C for 2h. Transform according to the method in Example 2.

4. Double enzyme digestion identification of recombinant plasmids

Obtain the recombinant plasmid according to the method in 1, respectively digest the recombinant plasmid with the corresponding fast cutting enzyme, and perform nucleic acid gel electrophoresis to confirm that the target gene has been connected to the expression vector. Take 400 μL of the confirmed bacterial liquid and send it to Sangon Biotechnology Co., Ltd. for sequencing.

As identified by nucleic acid gel electrophoresis, the recombinant plasmid can be enzymatically cut out a fragment of the size of the target gene, which is the same as the expected result (Figure 2). The recombinant Di-sHSP12.6 plasmid was transferred into BL21 Escherichia coli, and after screening on AMP-resistant plates, a single colony with regular morphology was picked for colony PCR identification, and the positive bacteria were sent to Sangon Biotechnology Co., Ltd. for sequencing. The target gene sequence was the same, indicating that the construction of the expression vector was successful.

Embodiment 4: the prokaryotic expression of protein

(1) Expand the bacterial liquid sequenced correctly in Example 2, and obtain the recombinant plasmid according to the method in Example 3.

(2) Take 10 μL of the plasmid and place it in a 1 mL EP tube, add 50 μL of LE.coliBL21 competent cells, mix gently with a micro-pippet, ice-bath for 30 minutes, and then bathe in water at 42°C for 90 seconds, then ice-bath for 5 minutes. Add 600μL LB liquid culture solution to the EP tube, incubate at 37℃, 180r/min for 1.5h, take 150μL bacterial solution and evenly inoculate it on the solid medium containing ampicillin (AMP) resistance, and incubate in a constant temperature incubator at 37℃ Cultured in medium for 12h.

(3) Carry out PCR identification according to the method in Example 2, conduct 5 mL expansion culture of the bacteria liquid identified as positive by PCR, 37 ° C, 180 r/min culture to OD value = 0.6, add inducer IPTG (1 mmol/L) to induce 6 h , another bottle without IPTG as a blank control.

(4) Take 1 mL of induced bacterial solution, centrifuge at 4°C, 12000 r/min for 1 min, collect bacterial pellet, add 40 μL ddH2O and 10 μL 5×SDS loading buffer and mix well.

(5) After heating in boiling water for 10 minutes, centrifuge at 12000 r/min at 4°C for 1 minute, and take an appropriate amount of supernatant for SDS-PAGE.

(6) Remove the PAGE gel, put it into a container with Coomassie Brilliant Blue staining agent, stain for about 30 minutes, decolorize in boiling water for 10 minutes, and observe the results under the gel imaging system.

SDS-PAGE showed that about 30 KDa was obtained (Di-sHSP12.6 was about 12 KDa in size, and the His tag was about 18 KDa in size) (Fig. 3).

Embodiment 5: Solubility analysis of recombinant protein

The bacteria capable of successful expression were inoculated in 1 LLB liquid medium (containing AMP 100 μg/mL), cultured at 37°C, 180 r/min for 3 h, and when OD=0.6, 10 mL IPTG was added for induction for 6 h. Centrifuge all the bacterial liquid at 12000r/min at 4°C for 10min, discard the supernatant and keep the remaining bacterial cells, lyse the cell wall with 10mL (50mmol/L) Tris-HCl, then put the suspension into the ice-water mixture, and ultrasonically disrupt the cells . 4°C, 12000r/min, centrifuge for 10min, take the supernatant and prepare samples according to the method in Example 4. The precipitates were respectively dissolved with gradient urea, and then the supernatants were collected by centrifugation respectively, and samples were prepared according to the method in Example 4. After the obtained samples were subjected to SDS-PAGE, they were imaged on a gel imaging system. The results showed that rDi-sHSP12.6 protein was expressed in the supernatant (Fig. 4).

Embodiment 6: Purification of recombinant protein

The bacteria capable of successful expression were inoculated in 1 LLB liquid medium (containing AMP 100 μg/mL), cultured at 37°C, 180 r/min for 3 h, and when OD=0.6, 10 mL IPTG was added for induction for 6 h to obtain recombinant protein bacterial liquid.

(1) Centrifuge all the bacterial liquid at 4°C, 12000r/min for 10min, discard the supernatant, and keep the bacterial cells.

(2) Lyse the cell wall with 10 mL (50 mmol/L) of Tris-HCl, then put the suspension into ice-water mixture, and ultrasonically disrupt the cells.

(3) 4°C, 12000r/min, centrifuge for 10min, and collect the supernatant.

(4) The precipitate was washed with 2 mol/L, 4 mol/L, 6 mol/L, 8 mol/L urea solution respectively, centrifuged at 12000 r/min at 4°C for 10 min, and the supernatant was collected.

(5) Filter the supernatant obtained above with a 0.45 μm filter membrane, and place it in a mixture of ice and water for later use.

(6) Filter BandingBuffer and ElutionBuffer with 0.22 μm filter membrane, put them in ice-water mixture together with 20% alcohol for later use, and remove air bubbles by ultrasonication for 1-2 minutes before use.

(7) Take out the nickel ion affinity chromatography column, connect it to the protein purification instrument, and equilibrate 5-10 bed volumes with BandingBuffer.

(8) Inject the protein, wait until the BandingBuffer is balanced for 5-10 bed volumes, start to elute with 13%, 25%, 50%, 75% and 100% ElutionBuffer (containing 400mM imidazole) respectively, and wait until the elution peak appears, Begin collecting protein samples.

(9) Perform ultrafiltration of the recombinant protein according to the method of using the ultrafiltration tube, during which time, add 0.22 μm membrane filter and sterilized PBS to replace imidazole and other substances.

(10) For the protein after ultrafiltration, use an ultra-micro ultraviolet spectrophotometer to measure the protein concentration, add 1-2 drops of glycerol and store at -80°C to ensure the stability of the protein.

The results showed that rDi-sHSP12.6 (Figure 5, lane 4) had a good purification effect.

Example 7: Analysis of recombinant protein immunogenicity and reactogenicity

2.7.1 Preparation of rabbit anti-recombinant protein IgG

Add an equal amount of Freund's incomplete adjuvant or Freund's complete adjuvant to the purified protein, so that the final protein concentration is 0.2 mg/mL, sonicate in a sonicator to a water-in-oil state emulsion (drop into water 1min without diffusion). The New Zealand rabbits were subcutaneously immunized in 4 times, and the specific immunization procedures are shown in Table 5:

table 5

Seven days after the fourth immunization, blood was collected from the vein of the rabbit's ear, and the serum was separated and stored at -20°C.

2. Rough extraction of IgG

(1) Take 10mL of serum and add 10mL of normal saline, then add 5mL of (NH ₄ ) ₂ SO ₄ saturated solution drop by drop (until flocculent precipitation occurs), making it a 20% (NH ₄ ) ₂ SO ₄ solution, while adding While stirring, let stand for 30min

(2) 4°C, 3000r/min, centrifuge for 20min, discard the precipitate (remove fibrin).

(3) Add about 15 mL of ( _NH4 )2SO4 saturated solution to the supernatant to make it a 50% ( _NH4 ) _2SO4 solution, and let it stand for 30 minutes.

(4) 4°C, 3000r/min, centrifuge for 20min, discard the supernatant.

(5) After the precipitate is fully dissolved with A2BangdingBuffer, it can be purified on the column.

3. Purification of IgG

(1) Install the purification column on the instrument according to the correct steps, and rinse the instrument tube with the first 20% alcohol until it is balanced.

(2) Equilibrate the column with A2BangdingBuffer until the ion line is balanced.

(3) Inject the crudely extracted IgG into the instrument, and continue to equilibrate the column with A2BangdingBuffer until the ion line is balanced.

(4) Then eluted with BBuffer. After the elution peak appeared, the flow rate was controlled to be low, and the purified IgG sample was collected, neutralized with Tris immediately, and the pH was about 8.0.

(5) Repeat the sample addition and purification, and repeat the steps (2)-(4).

(6) After purification, fill the instrument tube with 20% alcohol until the ion line is balanced.

(7) Take a small amount of purified IgG and prepare samples according to the method in Example 4 for verification.

Rabbit anti-r-Di-sHSP12.6 (Figure 5, lane 3) protein IgG was prepared according to the above method, and the rabbit anti-recombinant protein IgG was purified by nickel column affinity chromatography, the effect was good, and a heavy protein of about 50KDa appeared respectively. chain and light chain around 25KDa.

4. Western blot of Di-sHSP12.6 protein

(1) Subject the recombinant protein to SDS-PAGE.

(2) Cut off the excess part of the gel, cut 24 layers of filter paper (2 sheets) and nitrocellulose membrane (NC membrane) respectively, and place them together in the transfer buffer to equilibrate for 3 times, each time for 5 minutes.

(3) Stack the balanced gel, filter paper and NC membrane in the order of negative electrode plate, filter paper, gel, NC membrane and filter paper, and remove excess air bubbles in the middle with a glass rod.

(4) Cover the anode electric plate, set the current according to 1mA/cm2, and transfer for 30 minutes in total.

(5) After the membrane transfer, the gel was taken out and stained with Coomassie Brilliant Blue, and the efficiency of membrane transfer was observed in a gel imaging system.

(6) Put the NC membrane into a container and wash it with TBST for 3 times, 5 min each time.

(7) Add 5% skimmed milk powder to the container to block for 2 hours, add primary antibody (Dyrofilaria immigatus positive serum or prepared rabbit anti-IgG), dilute 1:100, 4°C overnight.

(8) Discard the primary antibody, wash 3 times with TBST, 5 min each time, add secondary antibody (HRP-labeled rabbit anti-dog IgG), dilute according to PBS 1:2000, and incubate at room temperature for 2 h.

(9) Discard the secondary antibody, wash 3 times with TBST for 5 min each time, take out the NC membrane and place it in a clean container, and add freshly prepared diaminobenzidine (DAB) chromogenic solution dropwise onto the NC membrane.

(10) Immediately stop the reaction with double distilled water after the band appears, and take pictures in the gel imaging system.

The immunogenicity and reactogenicity of rDi-sHSP12.6 protein were analyzed according to the method mentioned above. Western blot results showed that r-Di-sHSP12.6 protein had good immunogenicity and reactogenicity. The rabbit anti-IgG prepared from the recombinant protein can recognize the recombinant protein (Figure 5, lane 6, indicated by the white arrow), and the recombinant protein can be recognized by the serum of dogs naturally infected with heartworm disease, but not by the serum of healthy dogs, indicating that the protein It has good reactogenicity and immunogenicity (Figure 5, lane 5, indicated by the white arrow).

5. Indirect immunofluorescence localization

(1) Slicing: Dirofilaria immigrates female adults fixed with 4% paraformaldehyde were embedded in paraffin and sliced (thickness: 4mm).

(2) Baked slices: put the slices in a constant temperature oven at 60°C for 2 hours.

(3) Dewax and hydrate the baked slices in the following order: xylene Ⅰ (7min), xylene Ⅱ (7min), 100% alcohol Ⅰ (3min), 100% alcohol Ⅱ (3min), 95% Alcohol (3min), 85% alcohol (3min), 75% alcohol (3min), distilled water (8min).

(4) Put the slices after dewaxing and hydration into sodium citrate buffer for antigen heat recovery, and heat above 95°C for 15 minutes. After cooling, wash the slices with PBS 3 times, 5min/time.

(5) Drop 3% H2O2 on the tissue, 37°C, 25min, wash 3 times with PBS, 5min each time.

(6) Add 5% BSA blocking solution dropwise to the tissue, room temperature, 45min.

(7) Discard the excess liquid, add rabbit anti-recombinant protein IgG (diluted 1:100), incubate overnight at 4°C in a wet box; wash 3 times with PBS, 4min each time.

(8) Add FITC-labeled goat anti-rabbit IgG (1:100 dilution) diluted with 0.1% Evan's blue, and incubate at 37°C for 1 hour in the dark.

(9) Wash with PBS 3 times, 4min/time.

(10) Mount the slices with an appropriate amount of glycerol buffer, and place the slices under a fluorescence microscope for observation and imaging.

The distribution of Di-sHSP12.6 in the cross-sections of female and male adult Dirofilaria immigatus was detected by indirect immunofluorescence staining. The results showed that sHSP12.6 was mainly distributed in the epidermis and intestine of female Dirofilaria imimaginae, and a small amount was distributed in muscle; Male Dirofilaria immigatus is mainly distributed in the epidermis and can be secreted into the intestine, and only a small amount is distributed in the muscle (Figure 6, A, B, C, D, where A and C are positive, and B and D are negative).

Example 8: Establishment of rDi-sHSP12.6 protein indirect ELISA method

1. Operation method

(1) Take a 96-well ELISA plate, dilute the recombinant protein (antigen) with the coating solution, add 100 μL/well of the diluted protein, and coat overnight at 4°C.

(2) Discard the protein solution, wash with PBST 3 times, 5min each time, shake and wash on a micro-oscillator (try to fill up, but do not cross holes).

(3) Add 5% skimmed milk powder, 250 μL/well, and incubate at 37° C. for 1.5 h.

(4) Wash with PBST 3 times, 5min/time.

(5) Serum diluted with PBS was added, 100 μL/well, incubated at 37° C. for 1 hour.

(6) Wash with PBST 3 times, 5min/time.

(7) Add HRP-labeled rabbit anti-dog IgG diluted in proportion with PBS, 100 μL/well, at 37° C., and incubate for 1 hour.

(8) Wash with PBST 4 times, 5min/time.

(9) Add the soluble one-component substrate TMB chromogenic solution to the 96-well ELISA plate, 100 μL/well, and incubate at room temperature for 20 minutes in the dark.

(10) Add 2 mol/L H ₂ SO ₄ , 100 μL/well, to stop the reaction.

(11) Place the 96-well plate on a microplate reader, and measure its OD value (λ=450nm).

The indirect ELISA reagents of this embodiment can be used to form a kit.

2. Condition optimization

(1) Determine the optimal antigen coating concentration and serum concentration: refer to the checkerboard titration method to set 6 antigen coating concentrations such as: (20.20 μg, 10.10 μg, 5.05 μg, 2.53 μg, 1.26 μg and 0.63 μg/well ) and 4 serum dilutions (1:20, 1:40, 1:80 and 1:160), after completing the test according to the method 2.8.1, measure the reading on the microplate reader, calculate the P/N value, and determine Optimal antigen coating concentration and serum concentration. The condition with the positive serum OD450 close to 1 and the maximum P/N was regarded as the best.

(2) Determine the best blocking solution: according to the best antigen coating concentration and serum concentration, use 5% skimmed milk powder and 5% BSA as the blocking solution respectively, measure the reading on the microplate reader after completing the test, and calculate P/N value to determine the best blocking solution. The condition with the positive serum OD450 close to 1 and the maximum P/N was regarded as the best.

(3) Determine the optimal primary antibody incubation time: according to the optimal antigen coating concentration, serum concentration and blocking solution, set up 4 unused incubation times (0.5h, 1h, 1.5h and 2h). Measure the reading on the microplate reader, calculate the P/N value, and determine the optimal primary antibody incubation time. The condition with the positive serum OD450 close to 1 and the maximum P/N was regarded as the best.

(4) Determine the optimal secondary antibody incubation concentration: according to the optimal antigen coating concentration, serum concentration and primary antibody incubation time, set up 5 different secondary antibody dilutions (1:1000, 1:2000, 1:3000 , 1:4000 and 1:5000), after completing the test, measure the reading on the microplate reader, calculate the P/N value, and determine the optimal secondary antibody incubation concentration. The condition with the positive serum OD450 close to 1 and the maximum P/N was regarded as the best.

(5) Determine the best color development time: according to the above optimal conditions, set up 5 different color development times (10min, 15min, 20min, 25min, 30min), measure the reading on the microplate reader after completing the test, and calculate Get the P/N value to determine the best color development time. The condition with the positive serum OD450 close to 1 and the maximum P/N was regarded as the best.

3. Determination of critical value

Carry out the test according to the optimized test conditions in 2.8.2, and use the cutoff value method to determine the critical value. The OD450 of 24 heartworm negative sera were measured, and the mean and standard deviation of all serum OD450 were calculated. Cutoff value = mean value + 3 times standard deviation, and 3 repetitions were set.

4. Determination of specificity and sensitivity

The test was carried out according to the optimized test conditions in 2, and the positive sera of Echinococcus canis granulosus (8 copies), the positive serum of canine cysticercosis (2 copies) and the positive serum of hookworm (8 copies) collected in this laboratory were carried out respectively. and Toxocara canis serum (18 copies) were tested for cross-reactivity. According to the critical value determined in 3, calculate the corresponding specificity and sensitivity, the calculation formula is as follows: specificity = number of true negative sera / (number of true negative sera + number of false positive sera) × 100%, sensitivity = true positive Number of sera/(number of true positive sera + number of false negative sera)×100%, 3 replicates were set for each group.

According to the optimized indirect ELISA method, rDi-sHSP12.6 protein was used to detect Echinococcus canis positive sera (8 copies), canine cysticercosis positive sera (2 copies), hookworm positive sera (8 copies) and canine Toxocara canis serum (18 parts), the results showed that there was no cross-reaction with Echinococcus granulosus positive serum, Cysticercus canis positive serum and Hookworm positive serum. Only 2 copies had slight cross-reactivity (Fig. 7), and the specificity was 94.4% (34/36).

5. The repeatability of the detection test

(1) Intra-batch repeatability test: Coat the same concentration of recombinant protein in the same 96-well ELISA plate, detect 6 confirmed positive sera for heartworm disease, and repeat 3 wells for each serum, according to the optimized The ELISA method was used to conduct repeated experiments within the batch, calculate the coefficient of variation, and test the repeatability within the batch.

(2) Repeatability test between batches: 3 96-well ELISA plates were coated with the same concentration of recombinant protein, and 6 confirmed positive sera of heartworm disease were detected, and each serum was repeated for 3 wells, according to the optimized A good ELISA method, repeated experiments between batches, calculation of coefficient of variation, and test of repeatability between batches.

The coefficient of variation between batches is (5.63%-8.15%), and the coefficient of variation within batches is (0.96%-3.89%). The coefficient of variation between batches is less than 10%, and the coefficient of variation within batches is less than 5%, showing that the test has good repeatability .

6. Clinical testing

The established indirect ELISA method was used to detect 24 positive sera of Dirofilariasis and 24 negative sera of Dirofilariasis, and the reliability of the ELISA method was judged according to the critical value, specificity and sensitivity.

According to the optimized indirect ELISA method, rDi-sHSP12.6 protein was used to detect 24 Dirofilaria immature positive sera and 24 Dirofilaria immature negative sera, the results showed that 22 positive sera had OD450> critical value, 2 positive sera and If OD450 of 24 negative sera was less than the critical value (0.699), the sensitivity of this method was estimated to be 91.6% (22/24) (Figure 8).

The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

sequence listing

<110> Sichuan Agricultural University

<120> An ELISA kit for diagnosing heartworm disease

<130> MP1908394

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<170> SIPOSequenceListing 1.0

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<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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1 5 10 15

Trp Asp Trp Pro Leu Gln His Asn Asp Asp Val Val Lys Val Thr Asn

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Thr Asn Asp Lys Phe Glu Val Gly Leu Asp Ala Ser Phe Phe Thr Pro

35 40 45

Lys Glu Ile Glu Val Lys Val Cys Gly Asp Asn Leu Val Ile His Cys

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Arg His Glu Thr Arg Thr Asp Gln Tyr Gly Glu Ile Lys Arg Glu Ile

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Ser Arg Thr Tyr Lys Leu Pro Ser Asp Val Asp Thr Lys Thr Leu Thr

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Ser Asn Leu Thr Lys Arg Gly His Leu Val Ile Ala Ala Lys Lys Lys

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Ala

Claims (9)

1. An ELISA kit for diagnosing heartworm disease, characterized in that it comprises a solid phase carrier coated with heart shock protein of heartworm. 2. The ELISA kit according to claim 1, further comprising one or more of enzyme-labeled secondary antibodies, washing solution, chromogenic solution, blocking solution, diluent and stop solution. 3. The ELISA kit according to claim 2, wherein the enzyme-labeled secondary antibody is HRP-labeled rabbit anti-dog IgG. 4. The ELISA kit according to claim 2, wherein the washing solution is a PBS-T washing solution. 5. according to the described ELISA kit of claim 2, it is characterized in that, described chromogenic liquid is TMB chromogenic liquid. 6. The ELISA kit according to claim 2, wherein the blocking solution is skimmed milk. 7. The ELISA kit according to claim 2, wherein the diluent is PBS. 8. The ELISA kit according to claim 2, wherein the stop solution is a sulfuric acid solution. 9. The ELISA kit according to claim 1, wherein the sequence of the Dirofilaria imimaginum small heat shock protein is as shown in SEQ ID NO:1.