CN110208553B

CN110208553B - Application of taenia multiceps Antigen B

Info

Publication number: CN110208553B
Application number: CN201910561192.6A
Authority: CN
Inventors: 杨光友; 刘俞辰; 古小彬; 谢跃
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2019-06-26
Filing date: 2019-06-26
Publication date: 2020-11-20
Anticipated expiration: 2039-06-26
Also published as: CN110208553A

Abstract

The invention relates to the technical field of biology, and discloses a series of related applications of taenia multiceps Antigen B as a diagnosis Antigen of coenurosis cerebralis and the like, wherein related experimental results show that the taenia multiceps Antigen B can be recognized by goat serum infected with coenurosis cerebralis and does not react with negative serum, and the taenia multiceps Antigen B has good immunogenicity and reactogenicity; meanwhile, the kit shows higher sensitivity and specificity in an indirect ELISA method, and has better thermal stability compared with other proteins, and various results prove that the taenia multiceps Antigen B can be used as a diagnosis Antigen of the coenurosis cerebri and applied to a detection kit.

Description

Application of taenia multiceps Antigen B

Technical Field

The invention relates to the technical field of biology, in particular to application of taenia multiceps Antigen B.

Background

Coenurosis (Cerebral coenurosis) is a parasitic disease which is seriously harmful to cattle and sheep and is caused by middle-Taenia larvae of Taenia multiceps. The cercaria cerebri mainly parasitizes in the central nervous system of cattle and sheep, and also parasitizes in subcutaneous or muscular tissues, and human beings can also serve as intermediate hosts under the condition of eating taenia polycephala eggs by mistake. The disease often leads to the direct death of cattle and sheep, causing enormous economic losses to the livestock industry in europe, the usa, africa and asia, and accurate diagnosis of infected animals is one of the key directions of current research.

Diagnostic methods for coenurosis include anatomical, imaging and laboratory diagnostics. With the development of scientific technology, Polymerase Chain Reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) are also gradually applied to the clinical diagnosis of the disease. In recent years, good detection effects have been reported in ELISA diagnostic methods based on recombinant antigens of Taenia multiceps (Tm-P2, Tm-HSP70, Tm-GP50, Tm-GST and Tm-HSP60), but the thermal stability of the antigen used for production has been desired to be improved.

Disclosure of Invention

In view of the above, the invention aims to provide a taenia multiceps antitigen B (Tm-AgB) which is used as a diagnosis Antigen of coenurosis cerebralis and an application thereof in preparation of the diagnosis Antigen of coenurosis cerebralis, so that the taenia multiceps antitigen B has high specificity and sensitivity;

the invention also aims to provide the application of the taenia multiceps antitigen B (Tm-AgB) in the preparation of the kit for detecting the cerebral coenurosis, so that an ELISA method established by the taenia multiceps antitigen B shows higher specificity and sensitivity and can be used for ELISA detection;

in this context, Taenia multiceps Antigen B may be non-natural, e.g., synthetic or expressed from an artificial vector (often referred to in the art as recombinant protein rTm-AgB). The term "non-natural" means that the target substance is not naturally occurring in nature, which does not preclude the non-natural substance from having the same structure and/or composition as the naturally occurring substance.

Antigen B (Antigen B) is a lipoprotein which is peculiar to tapeworm, Monteriro et al find that molecules of 3 subunits AgB8/1, AgB8/2 and AgB8/3 of AgB can spontaneously polymerize into oligomers of 120-120 kDa, and the oligomers are stable to heat and have spiral circular dichroism spectrum characteristics and are similar to natural Ag B. The prior research shows that Antigen B has good thermal stability relative to other proteins of taenia multiceps, but the related research of Antigen B in the cerebral coenurosis has not been reported yet.

The recombinant taenia multiceps Antigen B (rTm-AgB which has completely the same amino acid sequence as Tm-AgB and is shown in SEQ ID NO: 1) is obtained by prokaryotic expression, and the construction of immunoblotting, ELISA and diagnosis method is carried out on the recombinant taenia multiceps Antigen B. The immunoblotting result shows that the recombinant antigen can be recognized by goat serum infected with cercaria cerebri to generate a specific strip, and negative serum of a control group has no reaction, which indicates that the recombinant antigen has stronger immunoreactivity and good immunogenicity.

The indirect ELISA result shows that the ELISA method established by rTm-AgB has the sensitivity as high as 95.8% (23/24) and the specificity as 87.5% (21/24), and has cross reaction with 3 parts of serum in each of Monizzschia taenia, Fasciola hepatica and Haemonchus contortus, 2 parts of serum in Zoacyta and 8 parts of serum in Echinococcus granulosus. rTm-TPx compared therewith was only 75.0% less sensitive (18/24) and 91.7% specific (22/24); rTm-HSP70 had a sensitivity of 87.5% (21/24) and a specificity of 83.3% (20/24); rTm-GST had a sensitivity of 83.3% (20/24) and a specificity of 91.7% (22/24); in conclusion, the rTm-AgB has higher sensitivity and specificity, better thermal stability compared with other proteins and better diagnosis effect, and is suitable to be used as a diagnosis antigen of the cercaria cerebralis and a related detection kit prepared by the diagnosis antigen.

Based on the content, the invention provides the application of taenia multiceps Antigen B as the diagnosis Antigen of the coenurosis cerebralis and the application in preparing the diagnosis Antigen of the coenurosis cerebralis. Meanwhile, the invention also provides the application of the taenia multiceps Antigen B in the preparation of the kit for diagnosing the cerebral coenurosis; among them, the kit is preferably an ELISA kit, and more specifically, the ELISA kit is a kit based on an ELISA indirect method.

According to the technical scheme, the taenia multiceps antitigen B is used as a diagnosis Antigen of the coenurosis cerebri and other related applications, and related experimental results show that the taenia multiceps antitigen B can be recognized by goat serum infected with the coenurosis cerebri and does not react with negative serum, so that the taenia multiceps antitigen B has good immunogenicity and reactogenicity; meanwhile, the kit shows higher sensitivity and specificity in an indirect ELISA method, and has better thermal stability compared with other proteins, and various results prove that the taenia multiceps Antigen B can be used as a diagnosis Antigen of the coenurosis cerebri and applied to a detection kit.

Drawings

FIG. 1 shows SDS-PAGE and immunoblot analysis of Tm-AgB; wherein, M: a protein standard; a: purified recombinant protein rTm-AgB; b: reacting the purified recombinant protein rTm-AgB with goat serum infected with coenurosis cerebralis; c: reacting the recombinant protein with negative goat serum;

FIG. 2 shows the sensitivity, specificity and cross-reactivity of the indirect ELISA method; the gray level line in the figure represents the cut-off value of the indirect ELISA method; statistical analysis was performed using SPSS version 20.0, and differences between different serogroups were counted using the mann-whitney U test method; a P value less than 0.05 indicates statistical significance.

Detailed Description

The invention discloses application of Taenia multiceps Antigen B, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the invention has been described in terms of embodiments, it will be apparent to those skilled in the art that the technology can be practiced and applied by modifying or appropriately combining the embodiments described herein without departing from the spirit and scope of the invention.

The invention amplifies Tm-AgB gene from cDNA by extracting total RNA of taenia multiceps imago, protocoid and oncosphere and reverse transcribing to cDNA. After T cloning, the amplified product is introduced into an expression vector in an enzyme digestion connection mode, and prokaryotic expression is carried out by using escherichia coli to obtain recombinant rTm-AgB.

The cysts of the cercaria were obtained from naturally infected goats in Sichuan province. Under aseptic conditions, the solution of the encapsulated capsule was extracted using a disposable syringe, centrifuged at 3000rpm for 5min, and then washed with sterilized physiological saline and PBS 3 times, respectively, to obtain the protocephalic segment. Taenia multiceps adult is obtained from 4 dogs 2 months old infected with 20000 metacercaria artificially for 30 days. The oncosphere is released after trypsinase treatment on the eggs in the fresh egg sections of the adult worms.

The application of Taenia multiceps Antigen B provided by the invention is further explained below.

Example 1: gene amplification

1. Extraction of taenia multiceps total RNA and cDNA synthesis

Taking out the taenia multiceps imago, the protocoid and the oncosphere preserved by liquid nitrogen, grinding the taenia multiceps imago, the protocoid and the oncosphere by using a mortar, and then extracting total RNA by referring to the specification of the RNA extraction kit of the animal tissue of the Tiangen. Total protein was extracted from adults with reference to the instructions of the solibao mammalian protein extraction kit.

2. Amplification of Taenia multiceps Tm-AgB gene

Primers for Tm-AgB reference transcriptome data Unigene17133, primers set with Primer Premier 5.0 software, all primers synthesized by Shanghai:

Tm-AgB upstream: 5'-CGGGATCCATGAAAGCCTACATTGTT-3' BamHI

Downstream of Tm-AgB: 5'-CCAAGCTTCTAGTTCTCCTCATCCAT-3' HindIII

The amplification procedure is shown in table 1:

TABLE 1

3. Cloning and sequencing of target genes

And (3) performing gel recovery on the PCR product according to the specification of a root gel recovery kit, connecting a target gene with a pMD19-T vector, transferring into DH5 alpha escherichia coli, and sequencing.

4. Amplification and basic physicochemical Properties of Tm-AgB Gene

A band of about 260bp is amplified by using cDNA of the taenia multiceps protocephalad knot as a template, and the homology of a sequence obtained by sequencing and a Tm-AgB (Unigene17133) sequence in a transcriptome reaches 100 percent.

The open reading frame in the Tm-AgB sequence comprises 261 encoded 86 amino acid polypeptides (the last 3 bases are stop codons, and do not encode amino acids). The signal peptide is predicted to occur at amino acids 1 to 20 of the sequence and appears to have a transmembrane region. The predicted subcellular localization of Tm-AgB indicates that the protein is a secreted protein.

5. Construction and identification of recombinant plasmids

After the clone sequencing identification is correct, respectively extracting pET-32a strain and plasmid of target gene, and using BamHI and HindIII quick-cutting enzyme to make double enzyme cutting, recovering enzyme-cut product. And connecting the product fragment with a pET-32a (+) expression vector, transforming and carrying out double enzyme digestion identification.

6. Expression and identification of recombinant plasmid in escherichia coli

6.1 expression and purification of recombinant proteins

The recombinant plasmid pET32a-Tm-AgB with correct sequencing is transferred into BL21(DE3) expression bacteria.

The expression bacteria are inoculated into two bottles of fresh LB (100 mu g/mL of AMP) culture solution containing 100mL, shake culture is carried out for 6h (160r/min) at 37 ℃, an inducer IPTG (1mmol/L) is added when the OD590 of the bacteria solution is 0.8, induction is carried out for 6h (160r/min) at 37 ℃, and the other bottle is not added with IPTG for control culture.

After the suspension was collected, 1.5mL was placed in two new EP tubes, centrifuged at 4 ℃ for 1min (12,000r/min) to collect the cells, 10. mu.L of 5 XSDS Buffer and 40. mu.L of PBS solution were added, and mixed well.

Boiling in water for 10min to break the thallus sufficiently, centrifuging at 4 deg.C for 10min (12,000r/min), and collecting the supernatant for SDS-PAGE.

And (4) staining for 1h by using Coomassie brilliant blue, and observing the expression condition after decoloring.

6.2 solubility analysis of expressed proteins

The expression strain containing the recombinant plasmid Pet32a-Tm-AgB was inoculated into 500mL of a liquid medium containing ampicillin, cultured at 37 ℃ (160rpm/min) to an OD590 of about 0.8, added with the optimum IPTG concentration, and induced for 6 hours.

The cell suspension was centrifuged for 10min (8000rpm), the supernatant was discarded, the pellet was suspended in a lysis solution (20mM Tris-HCl, pH 8.0), and the cells were disrupted by sonication.

Centrifuging the disrupted thallus lysate for 10min (12,000rpm) at 4 deg.C, and separating precipitate and supernatant; the precipitate was dissolved by adding an appropriate amount of 8M urea.

mu.L of each of the supernatant and the precipitate was taken, 10. mu.L of 5 XSDS gel loading buffer was added thereto, boiled for 10min, centrifuged for 10min (12,000rpm), and subjected to SDS-PAGE to determine whether the expression was soluble.

The target gene fragment is successfully connected to a pET-32a vector, and is transformed into BL21 escherichia coli for induced expression. The expression was maximal at 37 ℃ when induced with 1mM IPTG for 6 h. The expressed recombinant protein is about 26kDa in size (contains about 18kDa of the tag protein), and is in line with the expected size. The results of the solubility analysis showed that the protein was expressed as a soluble protein. The purified recombinant protein was a single band (FIG. 1).

Example 2: immunoblotting

The immunoreactivity of rTm-AgB and the serum of a sick animal is detected by immunoblotting, and the specific steps are as follows:

(1) after the protein electrophoresis is finished, the corresponding gel part where the protein is located is taken and put into a membrane transfer buffer solution for balancing for 3 times, and each time is 4 min.

(2) The nitrocellulose filter (NC membrane) and 24 layers of filter paper were soaked in transfer buffer for 5 min.

(3) The cathode electrode plate, 24 layers of filter paper, gel, NC membrane and 24 layers of filter paper are placed in a Bio-Rad semi-dry type transfer printing tank in sequence, and the anode electrode plate is covered.

(4) The electrotransfer device was attached to the electrotransfer instrument and transferred for 30min by adding 35mA of transfer buffer.

(5) After the transfer was completed, the NC membrane was taken out, soaked in TBST with 5% skim milk powder, and sealed overnight at 4 ℃.

(6) After the sealing is finished, the NC membrane is cut, the negative and positive are separately placed, and 1:1000 dilution of primary antibody, room temperature after 2h incubation, pour off the primary antibody, use TBST rapid washing membrane 3 times, 5 min/time.

(7) Diluting HRP-labeled goat-anti-rabbit or rabbit-anti-goat IgG according to a ratio of 1:1000, adding an NC membrane, incubating at room temperature for 2h, pouring out the secondary antibody, and rapidly washing the membrane for 3 times and 5 min/time by using TBST.

(8) The NC membrane was placed in a dish and washed with fresh substrate developing solution until development.

(9) After development, the NC membrane was washed with double distilled water to terminate development, and the results were recorded by photographing.

The result shows that the recombinant antigen can be recognized by the goat serum infected with the cercaria cerebri to generate a specific strip, while the negative serum of the control group has no reaction, which indicates that the recombinant antigen has stronger immunoreactivity and good immunogenicity (figure 1).

Example 3: establishment of Indirect ELISA method

1. Indirect ELISA procedure

(1) Diluting with antigen coating solution according to a ratio, and adding 100 mu L of antigen coating solution into a 96 enzyme label plate for coating in each hole;

(2) pouring out the coating liquid, patting off the liquid in the holes, washing with PBST for 5min each time, and repeating for four times;

(3) adding a sealing liquid for sealing.

(4) After washing, the serum was diluted in PBS in proportion, 100. mu.L of each well was incubated in an enzyme-labeled well, and the liquid was decanted off.

(5) After washing, 100. mu.L of diluted goat or sheep anti-rabbit secondary antibody labeled with HRP was added to each well and incubated.

(6) Adding a soluble single-component substrate TMB into the hole under the condition of keeping out of the sun for color development reaction;

(7) the reaction was stopped by adding 100. mu.L of 2M H2SO4 to the well, and the OD value was measured at an ultraviolet absorbance of 450 nm.

The indirect ELISA reagent according to the present embodiment may constitute a kit.

2. Condition optimization

(1) The optimal antigen and serum dilution concentration is determined by chessboard titration, 6 antigen concentration gradients are set, the serum is diluted from 1:20 to 1:640 in a multiple ratio, and the condition of maximum P/N is taken as the optimal condition.

(2) The optimal blocking solution was determined by blocking with 1% BSA, 5% BSA, 1% skim milk, and 5% skim milk, respectively, under the condition of maximum P/N.

(3) Determination of reaction time of negative and positive sera, optimal incubation time of negative and positive sera was selected according to the determined conditions, and three groups of 0.5h, 1h and 1.5h were set at 37 ℃ with the concentration of P/N being the maximum as the optimal.

(4) The optimal concentration of the secondary antibody effect is determined by searching four dilution concentrations of 1:2000, 1:3000, 1:4000 and 1:5000 respectively, and the condition of maximum P/N is taken as the optimal condition.

The result of searching the optimal reaction condition by using indirect ELISA established by rTm-AgB shows that the optimal coating condition is 4 ℃ overnight, the optimal concentration of antigen is 1 microgram per hole, the optimal dilution concentration of serum is 1:640, the optimal sealing solution and sealing condition is 5% skimmed milk powder sealed for 1 hour at 37 ℃, the optimal time for incubating serum is 37 ℃ for 1 hour, the optimal time for incubating secondary antibody is 37 ℃ for 1.5 hours, and the optimal dilution concentration of secondary antibody is 1:2000, the optimal condition for developing the color of a substrate is 37 ℃ for 15 minutes, under the above conditions, the P/N value of the negative and positive serum is measured to be the highest and is 2.23.

3. Determination of a threshold value

The OD450 of the 24 goat coenurosis negative sera was determined under optimal conditions. Three replicates were set up. Calculated as the cut-off value as mean +3 times standard deviation. The cutoff value of rTm-AgB was found to be 0.309, and the in-plate and inter-plate coefficient of variation was less than 10% for all indirect ELISA methods.

4. Specificity, sensitivity and cross-reactivity assays

The 24 goat sera confirmed to have cercaria were examined for sensitivity by dissection using an established indirect ELISA. The sensitivity calculation formula is: ELISA results Positive score 100/actual positive score.

The specificity of 24 goat sera dissected to confirm absence of parasitic infection was examined by established indirect ELISA. The specific calculation formula is as follows: ELISA results negative fraction × 100/actual negative fraction. Meanwhile, cross-reaction tests were carried out using 60 serum samples from sheep serum confirmed to be infected with echinococcus granulosus (12 parts) and moniz tapeworm (12 parts) by dissection and goat serum confirmed to be infected with Zona vesicularis (12 parts), Fasciola hepatica (12 parts) and Haemonchus contortus (12 parts), respectively.

The ELISA method established by rTm-AgB has the sensitivity as high as 95.8% (23/24) and the specificity as 87.5% (21/24), and has cross reaction with 3 serum of each of Monizzs tapeworm, Fasciola hepatica and Haemonchus contortus, 2 serum of Zoacyta and more serious cross reaction with Echinococcus granulosus (8 parts). rTm-TPx compared to them was only 75.0% less sensitive (18/24) and 91.7% specific (22/24); rTm-HSP70 had a sensitivity of 87.5% (21/24) and a specificity of 83.3% (20/24); whereas rTm-GST had a sensitivity of 83.3% (20/24) and a specificity of 91.7% (22/24), the cross-reactivity did not differ significantly from that described above (FIG. 2). Considering the overall data situation, rTm-AgB has higher sensitivity and specificity, better thermal stability compared with other proteins and better diagnostic effect, and is suitable to be used as a diagnostic antigen of the cercaria cerebralis and a related detection kit prepared by the diagnostic antigen.

5. In-batch and inter-batch repeatability tests

Taking the coated plates in the same batch, detecting 3 parts of goat serum which is determined to be positive by cercaria cerebralis through dissection, arranging 3 repeated holes in each part, performing batch repeated tests according to the established ELISA method, calculating the coefficient of variation, and detecting the batch repeatability of the method.

Taking 3 batches of coated plates, detecting 3 batches of cerebral coenurosis positive serum under the optimal condition, setting 3 repeat holes for each batch, performing batch-to-batch repeat tests according to the established ELISA method, calculating the coefficient of variation, and detecting the batch-to-batch repeatability of the method.

The inter-batch coefficient of variation is less than 10%, and the intra-batch coefficient of variation is less than 5%, indicating that the test has good repeatability.

6. Clinical testing

100 goats from 5 towns in Sichuan province were investigated for cercaria infections and each sample was tested in triplicate, and 18 sera were judged positive by detection of corresponding antibodies.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Sichuan university of agriculture

<120> application of Taenia multiceps Antigen B

<130> MP1913050

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 86

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Lys Ala Tyr Ile Val Leu Ala Leu Ala Leu Val Ala Phe Val Ala

1 5 10 15

Val Ala Arg Ala Glu Glu Asp Ile Glu Ser Lys Ala Arg Asp Gly Val

20 25 30

Met Lys Ser Leu Ala Glu Leu Lys Asp Phe Phe Lys Asn Asp Pro Met

35 40 45

Gly Gln Lys Leu Ala Ser Ile Cys Lys Asp Leu Lys Asp Leu Phe Leu

50 55 60

Met Ala Lys Thr Lys Thr His Ser Ala Phe Asn Asp Tyr Ile Lys Arg

65 70 75 80

Leu Met Asp Glu Glu Asn

85

Claims

1. The application of taenia multiceps Antigen B in preparing a cerebral coenurosis diagnosis Antigen; the sequence of the Antigen B protein is shown in SEQ ID NO. 1.

2. The application of taenia multiceps Antigen B in the preparation of a kit for diagnosing coenurosis cerebri; the sequence of the Antigen B protein is shown in SEQ ID NO. 1.

3. The use according to claim 2, wherein the kit is an ELISA kit.

4. The use according to claim 3, wherein the ELISA kit is a kit based on an ELISA indirect method.