阅读说明：本技术 用于检测早期旋毛虫病的特异性抗原及应用 (Specific antigen for detecting early trichinosis and application thereof ) 是由 杨娜 桑晓宇 王彦虎 丁莹莹 冯颖 陈冉 于 2021-05-26 设计创作，主要内容包括：本发明公开了用于检测早期旋毛虫病的特异性抗原及应用,用于检测早期旋毛虫病的蛋白质,包括第一蛋白质和第二蛋白质,所述第一蛋白质为如下(a1)或(a2)的蛋白质：(a1)氨基酸序列如SEQ ID.1所示的蛋白质；(a2)将(a1)限定的蛋白质的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加,且可与绵羊旋毛虫病血清抗体特异性结合的蛋白质。本发明用于检测早期旋毛虫病的特异性抗原及应用,首次将检测时间提前至48h,操作简便、敏感性和准确性良好。(The invention discloses a specific antigen for detecting early trichinosis and application thereof, and a protein for detecting early trichinosis comprises a first protein and a second protein, wherein the first protein is a protein (a1) or (a2) as follows: (a1) protein with amino acid sequence shown as SEQ ID.1; (a2) and (b) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of the protein defined in (a1) and can be specifically combined with the ovine trichinosis serum antibody. The specific antigen for detecting early trichinosis and the application thereof, the detection time is advanced to 48 hours for the first time, the operation is simple and convenient, and the sensitivity and the accuracy are good.)

1. A protein for detecting early trichinosis, comprising a first protein and a second protein, wherein the first protein is a protein of (a1) or (a2) as follows:

(a1) protein with amino acid sequence shown as SEQ ID.1;

(a2) a protein obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence of the protein defined in (a1), and capable of specifically binding to the ovine trichinosis serum antibody;

the second protein is a protein of (b1) or (b2) as follows:

(b1) protein with amino acid sequence shown as SEQ ID.3;

(b2) and (b) the amino acid sequence of the protein defined in (b1) is subjected to substitution and/or deletion and/or addition of one or more amino acid residues, and the protein can be specifically combined with the ovine trichinosis serum antibody.

2. A nucleic acid molecule encoding the first protein or the second protein of claim 1.

3. The nucleic acid molecule of claim 2, wherein the nucleic acid molecule is a gene encoding the first protein or the second protein of claim 1,

the gene encoding the first protein is a DNA molecule as described in any one of:

(c1) DNA molecule with the coding sequence shown in SEQ ID NO. 2;

(c2) a DNA molecule that hybridizes under stringent conditions to the DNA molecule defined in (c1) and encodes the first protein of claim 1;

(c3) a DNA molecule having more than 90% homology with the DNA molecule defined in (c1) or (c2) and encoding the first protein of claim 1;

the gene encoding the second protein is a DNA molecule as described in any one of:

(d1) DNA molecule with the coding sequence shown in SEQ ID NO. 4;

(d2) a DNA molecule that hybridizes under stringent conditions to the DNA molecule defined in (d1) and encodes the second protein of claim 1;

(d3) a DNA molecule having more than 90% homology with the DNA molecule defined in (d1) or (d2) and encoding the second protein of claim 1.

4. The use of the protein of claim 1 in the preparation of an immunochromatographic test strip for detecting trichinosis in sheep, wherein the test strip comprises a base plate, and a sample pad, a conjugate pad, a nitrocellulose membrane having a detection line and a quality control line, and a water absorbent pad, which are fixedly connected to the base plate in sequence, the detection line is close to one end of the conjugate pad, the quality control line is close to one end of the water absorbent pad, and the detection line is coated with the second protein of claim 1;

the conjugate pad is coated with a microsphere-labeled mouse IgG antibody and the first protein of claim 1;

and a goat anti-mouse IgG antibody is coated on the quality control line.

5. The use according to claim 4,

the first protein is a protein with 6 histidines added at the amino terminal of the amino acid sequence shown in SEQ ID.1;

the second protein is a protein with 6 histidines added at the amino terminal of the amino acid sequence shown in SEQ ID.3.

6. The use according to claim 4,

the coating concentration of the antigen protein at the detection line is 2 mg/mL.

7. The use according to claim 4,

in the conjugate pad, the concentration of the mouse IgG label is 1mg/mL, and the concentration of the first protein label is 50. mu.g/mL.

8. The use according to claim 4,

the coating concentration of the goat anti-mouse IgG is 2 mg/mL.

Technical Field

The invention relates to the technical field of biological detection, in particular to a specific antigen for detecting early trichinosis and application thereof.

Background

Trichinosis is a serious global zoonosis, and the human or ruminant mainly attacks diseases by eating the trichinosis infected meat by mistake, and the disease is a disease of two kinds of animals, which causes great economic loss to the animal husbandry every year. At present, regarding the diagnosis of trichina, the methods accepted by the international veterinary institute (OIE) are the tabletting microscopy and digestion methods, however, the two methods have certain holding time during detection, and the microscopy method is time-consuming, labor-consuming and low in sensitivity. Although the digestion method can improve the detection rate of the trichina to a certain extent, the detection rate of the trichina in the method is only required to be more than 3 worms in each gram of meat, and the method is extremely complicated in operation and needs a large amount of manpower and material resources. The immunological diagnosis, such as ELISA kit method, colloidal gold, etc., but the clinical application shows that the methods have low detection accuracy and low convenience, and cannot be convenient for basic workers to operate and detect. When animals are infected with trichina, the traditional diagnosis method is diagnosis at slaughter, however, in the slaughter process, the typical white needle point size of the cyst tissue is difficult to diagnose; serological diagnosis includes colloidal gold immunochromatography and enzyme-linked immunosorbent assay. These methods generally use excreta as an antigen, have high false positives, are expensive to prepare, and are difficult to commercialize.

In conclusion, the existing diagnosis means for trichina has the defects of complicated detection process, high false positive and high detection cost, so how to improve the immunodiagnosis of trichina and search for a reliable antigen is an urgent technical problem to be solved.

Disclosure of Invention

Therefore, the invention provides a specific antigen for detecting early trichinosis and application thereof.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention provides a protein for detecting early trichinosis, which comprises a first protein and a second protein, wherein the first protein is a protein (a1) or (a2) as follows:

(a1) protein with amino acid sequence shown as SEQ ID.1;

(a2) a protein obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence of the protein defined in (a1), and capable of specifically binding to the ovine trichinosis serum antibody;

the second protein is a protein of (b1) or (b2) as follows:

(b1) protein with amino acid sequence shown as SEQ ID.3;

(b2) and (b) the amino acid sequence of the protein defined in (b1) is subjected to substitution and/or deletion and/or addition of one or more amino acid residues, and the protein can be specifically combined with the ovine trichinosis serum antibody.

Nucleic acid molecules encoding the first protein or the second protein are also within the scope of the present invention.

Preferably, the nucleic acid molecule is a gene encoding the first protein or the second protein,

the gene encoding the first protein is a DNA molecule as described in any one of:

(c1) DNA molecule with the coding sequence shown in SEQ ID NO. 2;

(c2) a DNA molecule that hybridizes under stringent conditions to the DNA molecule defined in (c1) and encodes the first protein of claim 1;

(c3) a DNA molecule having more than 90% homology with the DNA molecule defined in (c1) or (c2) and encoding the first protein;

the gene encoding the second protein is a DNA molecule as described in any one of:

(d1) DNA molecule with the coding sequence shown in SEQ ID NO. 4;

(d2) a DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in (d1) and which encodes said second protein;

(d3) a DNA molecule having more than 90% homology with the DNA molecule defined in (d1) or (d2) and encoding the second protein of claim 1.

The invention also provides application of the protein in preparing an immunochromatographic test strip for detecting the trichinosis ovis, wherein the test strip comprises a bottom plate, and a nitrocellulose membrane and a water absorption pad which are sequentially and fixedly connected with a sample pad, a combination pad, a detection line and a quality control line on the bottom plate, the detection line is close to one end of the combination pad, the quality control line is close to one end of the water absorption pad, and the detection line is coated with the second protein as claimed in claim 1;

the conjugate pad is coated with a microsphere-labeled mouse IgG antibody and the first protein of claim 1;

and a goat anti-mouse IgG antibody is coated on the quality control line.

Preferably, the first protein is a protein with 6 histidines added at the amino terminal of the amino acid sequence shown in SEQ ID.1;

the second protein is a protein with 6 histidines added at the amino terminal of the amino acid sequence shown in SEQ ID.3.

Preferably, the coating concentration of the antigen protein at the detection line is 2 mg/mL.

Preferably, the concentration of mouse IgG label in the conjugate pad is 1mg/mL, and the concentration of the first protein label is 50. mu.g/mL.

Preferably, the coating concentration of the goat anti-mouse IgG is 2 mg/mL.

In the invention, the newborn larvae specific DNaseII-11 antigen is predicted by functional domains to be found to be a DNase II family protein, the homology of a plurality of proteins in the family is very high, and ko experiments further show that the protein plays an important role in the process of encystment formation and apoptosis of trichina. In recent years, researches show that DNase II family proteins are expressed in ML, NBL and Md periods of trichina, most of the DNase II family proteins are secreted proteins, can prevent the integration of exogenous genes and participate in functions such as invasion. At present, other functions and meanings of the trichina protein family are not clear, and further research is needed. The protein has 17B cell epitopes (critical value is 0.81) and 13T cell epitopes (critical value is 21). Good hydrophilicity, a signal peptide, no transmembrane, and is a secretory protein.

In the invention, the time-resolved fluorescence immunochromatography technology is utilized to combine the characteristics of immune labeling and immune chromatography. The immunochromatography test strip prepared by using the time-resolved fluorescent microspheres as the tracer can be used for detecting the trichina infected in cattle and sheep clinically, and has the advantages of simple operation, quick diagnosis, high sensitivity, strong specificity, and accurate qualitative determination, and wide application prospect.

The established method is based on a double-antigen sandwich immunochromatography method, and detects the trichinella antibody in the sample according to the specific reaction of the antigen and the antibody and the immunochromatography analysis technology. Recombinant antigens and mouse IgG antibodies marked by fluorescent microspheres are coated in a fluorescent binding pad (a glass fiber membrane), meanwhile, the recombinant antigens are coated on a detection line on a nitrocellulose membrane, and goat anti-mouse IgG antibodies are coated on a quality control line. During detection, if the sample contains trichina IgG antibody, the IgG antibody in the sample can be combined with the fluorescence-antigen in the binding pad at the front end of the test strip, flows along the lateral direction of the chromatographic strip due to siphonage, is captured by another recombinant antigen in the detection area to form a fluorescence-antigen-antibody-antigen immune complex, and the fluorescence intensity emitted by rare earth ions in the complex is measured by using a fluorescence immunoassay analyzer. Under the projection of an ultraviolet lamp (365nm), a test area and a control area of the chromatographic test strip excite the attached fluorescent microsphere conjugate, emitted light is gathered and converted into an electric signal, the strength of the electric signal is closely related to the number of fluorescent molecules, and the content of an analyte in a sample to be tested can be automatically analyzed by a fluorescence analyzer.

The invention has the following advantages:

the specific antigen for detecting early trichinosis and the application thereof, the detection time is advanced to 48 hours for the first time, the operation is simple and convenient, and the sensitivity and the accuracy are good;

the detection method established by the invention has good specificity, provides a rapid diagnosis method for epidemiological investigation and disease diagnosis, and can simultaneously detect early trichina-infected serum.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.

The structures, ratios, sizes, and the like shown in the present specification are only used for matching with the contents disclosed in the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions that the present invention can be implemented, so that the present invention has no technical significance, and any structural modifications, changes in the ratio relationship, or adjustments of the sizes, without affecting the effects and the achievable by the present invention, should still fall within the range that the technical contents disclosed in the present invention can cover.

FIG. 1 is a diagram of a double restriction enzyme digestion verification of a recombinant expression plasmid for reactive trypsin (Tsp _00436) -pET-28a according to an embodiment of the present invention, wherein the recombinant expression plasmid for reactive trypsin (Tsp _00436) -pET-28a is digested by 1-NdeI and XhoI; 2-reactive trypsin (Tsp _00436) -pET-28a recombinant expression plasmid;

FIG. 2 shows SDS-PAGE and Western-blot verification of a positive trypsin (Tsp _00436) -His recombinant protein according to an embodiment of the present invention;

FIG. 3 is a diagram of the double restriction enzyme digestion verification of the recombinant expression plasmid pET-28a-newborn specific DNaseII-11 provided in the example of the present invention, wherein 1-NdeI and XhoI is used to digest the recombinant expression plasmid pET-28 a-reactive tryptsin (Tsp _ 00436); 2-pET-28 a-reactive trypsin (Tsp _00436) recombinant expression plasmid;

FIG. 4 shows the SDS-PAGE and Western-blot verification of the newborn great specific DNaseII-11-His recombinant protein provided in the examples of the present invention;

fig. 5 is a schematic structural diagram of a time-resolved fluorescent microsphere chromatography test strip provided in an embodiment of the present invention, wherein the test strip comprises a 1-PVC base plate; 2-sample pad; 3-a conjugate pad; 4-absorbent paper; 5-nitrocellulose membrane; 6-detection line T; 7-quality control line C;

fig. 6 is a specific detection result of the time-resolved fluorescent microsphere chromatography test strip provided in the embodiment of the present invention, wherein the 1-epizoonosis positive serum; 2-sheep pratylenchus pratylencus positive serum; 3-sheep oesophageal oral nematode positive serum; 4-sheep woolly tail nematode positive serum;

fig. 7 is a graph of the sensitivity detection result of the time-resolved fluorescent microsphere chromatography test strip provided in the embodiment of the present invention, wherein the ratio of 1-4: the serum is sequentially mixed according to the following ratio of 1:400, 1:800, 1:1600, 1:3200, 5: a sample diluent;

FIG. 8 is a diagram showing the results of serum and negative serum tests performed by the test strip of the present invention for detecting trichinosis at different periods.

Detailed Description

The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

The present example provides a method for preparing a recombinant antigen of a reactive trypsin (Tsp _00436), wherein an amino acid sequence of the recombinant antigen of the reactive trypsin (Tsp _00436) optimized according to a gene sequence of the reactive trypsin (Tsp _00436) is shown in SEQ ID No.1, and a gene sequence encoding the protein is shown in SEQ ID No. 2. The amino acid sequence of the newborn larvae specific DNaseII-11 recombinant antigen is shown as SEQ ID NO.3, and the gene sequence for coding the protein is shown as SEQ ID NO. 4.

1. The gene fragment is synthesized by Suzhou Hongxi biotechnology GmbH with expression vector reactive trypsin (Tsp-00436) and newbornelvae-specific DNaseII-11 containing corresponding target genes, and the steps of double enzyme digestion are as follows:

(1) centrifuging the expression vector freeze-dried powder filled with the target gene at 3000 rpm/normal temperature for 1 min.

(2) With 50. mu.L sterile ddH₂Dissolving the freeze-dried powder by using a vortex instrument, gently mixing the mixture, and instantly separating the mixture for 30 seconds by using a palm centrifuge.

(3) The concentration was measured by NANODROP.

(4) The expression vector plasmid pET-28a was digested with restriction enzymes, and the digestion system is shown in Table 1. The reaction conditions were 37 deg.C/10 min.

TABLE 1 pET-28a expression plasmid double digestion system

2. Competence of recombinant plasmid transformation expression

(1) 50. mu.L of ice-cooled BL21(DE3) was placed in a 1.5mL sterile centrifuge tube, thawed (without being hand-rubbed) in an ice box, and 5. mu.L of pET-28 a-positive trypsin (Tsp-00436) and pET-28a-newborn large space specific DNaseII-11 expression plasmid were pipetted and allowed to stand on ice for 30 min.

(2) After ice-bath, the centrifuge tube was placed in a water bath, floated for 45S under thermal shock at 42 ℃, and then transferred to ice for 2min in ice-bath.

(3) 400-500. mu.L of sterile liquid LB medium was added to each tube at 220rpm in a sterile super clean bench and incubated for 1h at 37 ℃ on a shaker.

(4) The LB solid plate with corresponding resistance is preheated at 37 ℃, and is taken out for standby after the water vapor in the plate is completely volatilized.

(5) According to the experimental requirements, sucking a proper amount of transformed competent cells, dripping the competent cells in a preheated plate in a scattered manner, uniformly coating the competent cells, and putting the competent cells into a 37 ℃ incubator.

(6) The cells were incubated overnight in a 37 ℃ incubator.

3. Small-scale inducible expression of His-tagged recombinant proteins

(1) Single colonies of transformed BL21(DE3) were picked up with a sterile white pipette tip and, after correct PCR, inoculated into 10mL of liquid LB medium containing the corresponding resistance, incubated at 200rpm/37 ℃ overnight.

(2) The next day, after the bacterial preservation in the sterile operating platform, 100. mu.L of the bacterial liquid was pipetted and inoculated into 10mL of LB medium containing relatively resistant liquid, and cultured at 37 ℃ and 200rpm, when the OD of the bacterial liquid is_600nmWhen the concentration is 0.6-0.8, IPTG induction can be added.

(3) Before adding the protein inducer, 100. mu.L of uninduced whole bacteria were aspirated in a sterile operating table as a negative control. Then, 0.1M IPTG inducer was added to the mixture so that the final concentration of IPTG was 0.1mM, 0.2mM, and 0.5mM, and induction was carried out at different temperatures (16 ℃ C., 32 ℃ C., 37 ℃ C.).

(4) Collecting bacterial liquid which is cultured for 16h at a low temperature of 16 ℃, 8h at a low temperature of 32 ℃ and 5h at a constant temperature of 37 ℃, placing the collected bacterial liquid in a 15mL enzyme removing tube, centrifuging for 15min at 4000rpm of a 4 ℃ centrifuge, discarding supernatant, suspending thallus precipitates by using 1mL phosphate buffer solution, taking 60 muL of the bacterial liquid as a whole induced bacterial sample, crushing the rest bacterial liquid by using a small ultrasonic crusher, wherein the crushing power of the instrument is 28w, performing ultrasonic treatment for 2s, and stopping for 3s, observing the thallus to be clear, placing the crushed bacterial liquid in a 1.5mL EP tube, centrifuging for 10min at 5000rpm in a 4 ℃ centrifuge, taking 60 muL of the supernatant (supernatant after induction), discarding the supernatant, and suspending the precipitates by using 1mL phosphate buffer solution, and taking 60 muL as precipitate after induction.

(5) Adding the supernatant, precipitate and whole bacteria at different temperatures and different IPTG concentrations into 20 μ L1 xSDS-PAGE protein loading buffer respectively, boiling in 100 deg.C boiling water for 10min, and taking out.

(6) And (3) loading 10 mu L of prepared whole bacteria, supernatant and precipitated protein samples with different temperatures and IPTG concentrations (sequentially loading protein samples with the same temperature and different IPTG concentrations for subsequent observation) into 10% SDS-PAGE protein gel electrophoresis, wherein the setting power of a protein electrophoresis tank is as follows: voltage 150V and time 55 min.

(7) The cut protein gel was placed in a protein staining cassette into which G250-Coomassie brilliant blue staining solution was previously poured, and shaken on a horizontal shaker for 70 min.

(8) And (3) putting the dyed protein gel into a decoloring solution, decoloring overnight, observing the expression condition of the recombinant protein by using a gel irradiating instrument, and simultaneously determining whether the transferred expression competence target gene is expressed, the expression quantity and the expression form are soluble in supernatant or inclusion bodies in precipitate.

In the step, both proteins are expressed in inclusion bodies under small induction, the optimal induction temperature is 37 ℃, and the IPTG concentration of tsp-00436 is 0.2 mM. The IPTG concentration for inducing expression of newborne larvae specific DNaseII-11 was 0.5 mM.

4. Large-scale expression and purification of His tag inclusion body recombinant protein

(1) Taking out the bacterial liquid with the His-labeled recombinant protein from a refrigerator at the temperature of minus 20 ℃ in advance, putting the bacterial liquid into the refrigerator at the temperature of 4 ℃, picking a proper amount of bacterial liquid by using an inoculating loop in an aseptic ultra-clean bench after the bacterial liquid is completely melted, quickly marking a line on a Kana resistance culture plate by adopting a cross marking method, putting a culture dish into a constant-temperature incubator at the temperature of 37 ℃ and culturing for 10 hours.

(2) The next day, the petri dish was removed, a single colony of 1-2mm with a smooth morphology was picked with a white pipette tip, inoculated into 20mL Kana-resistant liquid LB medium, and cultured overnight in a constant temperature shaker at 37 ℃ at 200 rpm.

(3) In a sterile operating platform, 5mL of overnight-cultured bacterial liquid is sucked, added into 500mL of Kana-resistant liquid LB medium, shaken at 37 ℃ for 3h at 200rpm, and then the OD value of the bacterial liquid is measured.

(4) If the OD value of the spectrophotometer is between 0.6 and 0.8, the bacteria liquid is proved to reach the peak value of the growth curve, and the culture is carried out according to the condition that the protein is optimized when being induced and expressed in small quantity.

(5) And (3) dividing the shaken bacterial liquid into a plurality of sterile 50mL centrifuge tubes from the conical flask, centrifuging for 15min at 4000rpm in a low-temperature centrifuge, discarding the supernatant into a waste liquid tank, adding 5mL PBS into each tube, resuspending the precipitate by using a vortex device, collecting the precipitate into two sterile 50mL centrifuge tubes, centrifuging again under the same condition, adding 25mL PBS into each tube of bacterial liquid, resuspending and washing, centrifuging for 20min at 4000rpm in a 4 ℃ centrifuge, and discarding the supernatant.

(6) Setting a cooler to be precooled at 4 ℃ half an hour in advance, starting a high-pressure crusher, after air pressure buffering is finished, sequentially passing through a crushing pipe by using 75% alcohol, ddH2O and His Binding Buffer, and exhausting and discharging waste liquid after 3 times.

(7) Adding a proper amount of PBS according to the amount of the thallus precipitate, completely suspending the thallus precipitate by using a vortex device, pouring the thallus precipitate into a high-pressure crushing tube, repeatedly crushing for 3-4 times, centrifuging for 13min by using a superhigh speed 4 ℃ centrifuge at 12000rpm, and reserving the precipitate for later use.

(8) The precipitate was washed with 100. mu.L of ddH₂Mixing, adding 10mL of Binding Buffer containing 6M-8M urea, mixing and placing in a circular rotating instrument for sensing at room temperature overnight.

(9) The next day, the sensed disrupted bacterial liquid was centrifuged at 12000rpm for 13min in a 4 ℃ centrifuge and the supernatant was retained for later use.

(10) And (2) rinsing the empty purification column twice by using 6M-8M urea His Binding Buffer, reserving a small amount of His Binding Buffer, absorbing a proper amount of Ni-Agarose Resin according to the amount of supernatant, slowly adding the Ni-Agarose Resin into the purification column, opening a bottom switch when the Ni-beads completely sink to the bottom, and after waste liquid is discharged, passing through the column by using a proper amount of His Binding Buffer to wash the filler.

(11) And (4) adding the supernatant collected in the step (9) into the treated Ni-Agarose Resin filler, placing on a horizontal rotator, and slowly feeling for 3 hours at room temperature.

(12) And pouring the mixture of the combined supernatant and the Ni-Agarose Resin into a purification column, after the Ni-Agarose Resin is completely precipitated, opening a bottom switch, adjusting the flow rate, and finishing column passing when the Ni-Agarose Resin is completely in the purification column.

(13) In the first exploration of the purification conditions of the His recombinant protein, the elution is carried out by imidazole with low to high concentration: 6M-8M urea binding buffer, 10, 20, 40, 60, 80, 100, 250, 500mM, 3mL each time were eluted sequentially and collected in 2mL sterile centrifuge tubes and stored temporarily in ice boxes.

(14) Respectively preparing samples of protein eluted by imidazole with different gradients, column passing liquid and Ni-Agarose Resin eluted for the last time, performing protein gel electrophoresis, observing a target band by using a protein photographic instrument, and determining impurity washing and elution concentration of the protein.

In this step, the optimal imidazole elution concentration of Tsp _00436 protein was 250 mM. The optimal elution concentration of the newborne larvae specific DNaseII-11 protein was 500 mM.

5. Dialysis and concentration of His-tag inclusion body recombinant protein

(1) Observing SDS-PAGE protein gel, determining the volume of the target protein according to the thickness and purity of a band, and performing centrifugal concentration on the target protein by a concentrator at 1500rpm for 30min for later use.

(2) Will dialyse the one end of bag earlier, roll over two little book, clip one end with albumen dialysis clip, add purpose protein slowly along the edge of dialysis bag, roll over two little book with the other end dialysis bag again, press from both sides tightly, carry out gradient dialysis:

a. the proteins sandwiched between dialysis bags were dialyzed overnight in 3L PBS containing 6M urea and 0.5M arginine.

b. The next day, the dialyzed protein was observed for the presence of floc, and if floc was present, the dialysis was terminated, and if no floc was present, the entrapped target protein was placed in 2L PBS containing 4M urea and 0.5M arginine, and dialyzed for 4 hours, and the presence of floc in the dialysis bag was observed every 1.5 hours.

c. If no floc is present, the sandwiched protein is placed in 2L PBS containing 2M urea and 0.5M arginine, dialyzed for 4h, and observed every 1.5h for the presence of floc in the dialysis bag.

d. If no floc is present, the entrapped protein is placed in 3L PBS containing 0.5M arginine and dialyzed for 4h, and the presence of floc in the dialysis bag is observed every 1.5 h.

(3) Transferring the dialyzed target protein into a 2mL sterile centrifuge tube, placing the tube in an ice box, measuring the concentration of the protein by using Nanodrop, and observing the band of the target protein according to SDS-PAGE to determine whether the protein is concentrated or not.

(4) The denatured target protein is dispensed into sterile 200. mu.L centrifuge tubes and stored at-80 ℃ preferably immediately.

6. Western-blot identification of His tag inclusion body recombinant protein

(1) And performing SDS-PAGE gel running according to the third step.

(2) Cutting a PVDF film with proper size according to the size of the protein adhesive, activating for 1min by using methanol, cutting qualitative filter paper (3 layers are one) into proper size, soaking the filter paper in a film transferring solution to drive out bubbles between the filter paper and the filter paper, placing a black surface of a clamp below the film transferring solution in a horizontal manner, sequentially placing a water absorbing net above the filter paper, 3 layers of qualitative filter paper, the protein adhesive, the PVDF film, the qualitative filter paper and the water absorbing net above the filter paper, closing the clamp after no bubbles exist, placing the clamp into a film transferring groove, enabling the black surface of the clamp to face the black surface of the film transferring groove, enabling a transparent surface of the clamp to face a red surface of the film transferring groove, checking positive and negative electrodes, starting to transfer the film, and setting power as follows: current 175mA, time 86 min.

(3) After membrane transfer, the PVDF membrane was placed in a previously prepared plastic box containing blocking solution (5% skim milk), placed in a 37 ℃ incubator, and sealed for 1h on a shaker.

(4) Blocking with 5% skim milk containing His-tag monoclonal antibody (1:5000), and incubating overnight at 4 ℃.

(5) PBST membrane washing, each time for 10min, washing 4 times.

(6) AP goat anti-mouse IgG 1:5, 000PBST dilution, 37 degrees C were incubated for 60 min.

(7) And (5) repeating the step.

(8) Preparing a proper amount of BCIP/NET color developing reagent, performing dark color development for 2-3min, observing the result and scanning.

A recombinant expression plasmid of the target gene putative trypsin (Tsp _00436) is synthesized, the target gene is 1275bp, and is identified after being cut by NdeI and XhoI restriction enzymes, and the double-enzyme cutting verification result is shown in figure 1. The purified protein was confirmed by SDS-PAGE and immunoblotting to have the same size as the expected protein, and the recombinant protein size was 55kDa, as shown in FIG. 2.

Synthesizing a recombinant expression plasmid of a target gene newborn space specific DNaseII-11, wherein the target gene is 981bp, and is identified after being cut by NdeI and XhoI restriction enzymes, and the double-enzyme digestion verification result is shown in figure 3. The purified protein was confirmed by SDS-PAGE and immunoblotting to have the same size as the expected protein, and the recombinant protein size was 37kDa, as shown in FIG. 4.

Example 2

As shown in fig. 5, the present embodiment provides a trichinosis time-resolved fluoroimmunoassay test strip, which includes a sample pad 2, a binding pad 3, a nitrocellulose membrane 5(NC membrane, chromatographic membrane), a water absorbent paper 4 and a PVC base plate 1. A sample pad 2, a combination pad 3, a nitrocellulose membrane 5 and absorbent paper 4 on the PVC base plate 1. Wherein, one end of the nitrocellulose membrane 5 is laminated with one end of the combination pad 3, the other end of the nitrocellulose membrane 5 is laminated with one end of the absorbent paper 4, and one end of the sample pad 2 is laminated with the other end of the combination pad 3; the combination pad 3 is coated with goat anti-bovine IgG antibody marked by fluorescent microspheres, one side of the nitrocellulose membrane 5, which is close to the combination pad, is provided with a detection line 6, one side of the nitrocellulose membrane, which is close to the absorbent paper 4, is provided with a quality control line 7,

the sample pad 2 is provided with a sample adding hole, the combination pad 3 is coated with a mixture of 1mg/mL mouse IgG labeled by time-resolved fluorescent microspheres and 50 mu g/mL reactive trypsin (Tsp _00436) recombinant antigen, the detection line 6 of the nitrocellulose membrane 5 is provided with newborne large specific DNaseII-11 recombinant antigen, the quality control line 7 is coated with goat anti-mouse IgG, and the coating concentration of the goat anti-mouse IgG is 2 mg/mL.

In order to improve the accuracy of the detection of the sheep trichinosis time-resolved fluorescence immunochromatographic test strip, the invention gropes the following conditions:

the precondition for optimizing the time-resolved fluorescent microsphere immunochromatography test strip is as follows: other conditions are unchanged, changing only one of the variables at a time. The reaction conditions are as follows: the sample adding amount of the positive sample and the negative sample is 75 mu L (the ratio of serum to diluent is 1:2), and the reaction time is 15 min. The coating parameter is 0.75 mu L/cm, and the antigen or antibody is coated on the NC membrane to form a detection line (T) and a quality control line (C).

1. Determination of coating antigens

The newborn larvae specific DNaseII-11 recombinant antigen was coated at 1.0mg/ml, 2.0mg/ml, 3.0mg/ml working concentration, and the C-line goat anti-mouse IgG was coated at 2.0 mg/ml. Drying in an oven with the temperature of 45 +/-1 ℃ and the humidity of less than or equal to 35% for 16 h. The sample was prepared by matching the fluorescent pad and the sample pad. The value of T, C is read in a fluorescence immunoassay analyzer, T/C is calculated, and the optimal concentration of the coating antigen is determined. The conditions selected were: when a positive sample is dripped, the T/C value is maximum; when the negative sample was added dropwise, the T/C value was minimal.

The results are shown in table 1, wherein N represents a negative serum sample; p represents a positive serum sample. The optimal coating amount is obtained when the concentration of the newborne larvae specific DNaseII-11 recombinant antigen is 2.0 mg/ml.

TABLE 1 measurement results of coating antigen concentration

2. Determination of the concentration of the labeled antigen

Labeling different amounts of reactive trypsin (Tsp _00436) antigens by the same labeling method respectively by using the treated fluorescent microspheres, wherein the labeling amounts are respectively as follows: 25. mu.g/ml, 50. mu.g/ml, 100. mu.g/ml. The sample is diluted by 4 times of the dilution concentration to prepare a small sample, a coating sheet and a sample pad are matched, T, C values are read by a fluorescence immunoassay analyzer, and T/C is calculated to determine the optimal labeled antigen concentration. The conditions selected were: when a positive sample is dripped, the T/C value is maximum; when the negative sample was added dropwise, the T/C value was minimal.

The results are shown in Table 2, wherein N represents a negative serum sample; p represents a positive serum sample. The optimal conjugate pad labeling concentration was found when the labeling antigen reactive trypsin (Tsp _00436) concentration was 50. mu.g/ml.

TABLE 2 measurement results of labeled antigen concentration

3. Determination of dilution ratio of fluorescent labeling solution

Diluting the marked fluorescent microspheres by 2 times, 4 times and 8 times with 3 dilutions respectively. Preparing a small sample, matching with a coating sheet and a sample pad, reading an T, C value in a fluorescence immunoassay analyzer, and calculating T/C to determine the optimal dilution ratio of the fluorescence labeling solution. The conditions selected were: when a positive sample is dripped, the T/C value is maximum; when the negative sample was added dropwise, the T/C value was minimal.

The results are shown in Table 3, wherein N represents a negative serum sample; p represents a positive serum sample. When the dilution ratio of the fluorescent labeling solution is 4 times, the optimal dilution ratio is the optimal dilution ratio of the fluorescent labeling solution.

TABLE 3 measurement results of dilution ratio of fluorescent labeling solution

4. Determination of fluorescent working fluid

Selecting proper fluorescent microsphere suspension according to the determined dilution ratio of the fluorescent labeling solution, wherein the proper fluorescent microsphere suspension is divided into three groups: the first group is: 0.05m tris-HCl, 0.9% NaCl, 0.05% BSA, 0.05% Tween20, adjusted to pH 7.9; the second group is: 0.02m tris-HCl, 0.4% BSA, adjusted to pH 7.5; the third group is: 0.1M Tris-HCl, 0.1% BSA (5mL), 10% S9, 10% Tween20, 10% PEG12000, 3g trehalose. Preparing a small sample, matching with a coating sheet and a sample pad, reading T, C values in a fluorescence immunoassay analyzer, and calculating T/C to determine the optimal fluorescence working solution. The conditions selected were: when a positive sample is dripped, the T/C value is maximum; when the negative sample was added dropwise, the T/C value was minimal.

The results are shown in Table 4, wherein N represents a negative serum sample; p represents a positive serum sample. The second set of fluorescent working fluids was found to be the best fluorescent working fluid.

TABLE 4 fluorescent working solution test results

5. Determination of sample pad treatment fluid

Three sample pad treatment solutions were prepared in order to better bind the antibody in the sample to the antigen labeled with the fluorescent microspheres in the conjugate padIt is divided into three groups. The first group is: 1% BSA, 0.1% Triton-100, 0.02mol/L phosphate buffer pH 7.4; the second group is: 0.1M Na₂B₄O₇·10H₂O, 1% PVP, 0.2% Casein-Na, 1% Triton-X100, 1% Tetronic 1307, 0.2% NaN3, adjusted to pH 9.3; the third group is: 0.5M boric acid buffer, 1% triton x-100, 1% PVP, 2% NaCl, adjusted to pH 9.0. Preparing a small sample, matching with a coating sheet and a fluorescent pad, reading T, C values in a fluorescence immunoassay analyzer, and calculating T/C to determine the optimal sample pad treatment solution. The conditions selected were: when a positive sample is dripped, the T/C value is maximum; when the negative sample was added dropwise, the T/C value was minimal.

The results are shown in Table 5, wherein N represents a negative serum sample; p represents a positive serum sample. It was found that the second set of sample pad treatment solutions was the best sample pad treatment solution.

TABLE 5 test results of sample pad treatment solution

6. Determination of reaction time

Sucking 75 μ L of sample with a pipette, adding into 150 μ L of sample buffer, mixing well for 30s^-1And min, sucking 75 mu L of the mixed sample by using a pipette, dripping the sample into the sample adding hole of the detection card, and reading after reacting for 10min, 15min and 20min respectively. The value of T, C was read in a fluorescence immunoassay analyzer, and T/C was calculated to determine the optimal reaction time. The conditions selected were: when a positive sample is dripped, the T/C value is maximum; when the negative sample was added dropwise, the T/C value was minimal.

The results are shown in Table 6, wherein N represents a negative serum sample; p represents a positive serum sample. The optimal reaction time of the chromatographic test strip is 15 min.

TABLE 6 results of measurement of reaction time

7. Determination of sample buffer

To ensure that the antigen and antibody are fully reacted, three sample dilutions were searched, divided into three groups, each: a first group: 0.02M Tris-HCl pH 7.8, 0.9% NaCl, 0.1% BSA, 0.5% Tween-20, 0.1% NaN₃(ii) a Second group: 0.05M Tris-HCl pH 7.8, 0.9% NaCl, 1.5% BSA, 0.01% Tween-20, 0.1% NaN₃(ii) a Third group: PBS buffer pH 7.8. The value of T, C was read in a fluorescence immunoassay analyzer and the T/C was calculated to determine the optimal sample dilution. The conditions selected were: when a positive sample is dripped, the T/C value is maximum; when the negative sample was added dropwise, the T/C value was minimal.

The results are shown in Table 7, wherein N represents a negative serum sample; p represents a positive serum sample. The second set of sample buffers is the optimal sample diluent.

TABLE 7 test results of sample buffers

8. Determination of dilution ratio of sample

The sample and the sample buffer solution are mixed according to the proportion of the following table 8 respectively, the mixed sample is dripped into a sample adding hole of a detection card, T, C values are read by a fluorescence immunoassay analyzer, and T/C is calculated to determine the optimal sample dilution proportion.

TABLE 8 dilution ratio of sample to sample buffer

The results are shown in Table 9, wherein N represents a negative serum sample; p represents a positive serum sample. The optimal dilution ratio is 1:2 (sample volume: sample buffer)

TABLE 9 test results of sample dilutions

In this embodiment, in the time-resolved fluorescence immunochromatographic test strip for early diagnosis of trichinosis, the concentration of the newborne larvae specific DNaseII-11 recombinant antigen is 2.0mg/ml, the concentration of the labeled antigen reactive trypsin (Tsp _00436) is 50 μ g/ml, the dilution ratio of the fluorescent labeling solution is 4 times, and the fluorescent microsphere suspension is: 0.02m tris-HCl, 0.4% BSA, adjusted to pH 7.5; the sample pad treatment solution was 0.1M Na₂B₄O₇·10H₂O、1％PVP、0.2％Casein-Na、1％Triton-X100、1％Tetronic 1307、0.2％NaN₃Adjusting the pH value to 9.3; the optimal reaction time of the chromatographic test strip is 15 min. The sample buffer solution was 0.05M Tris-HCl pH 7.8, 0.9% NaCl, 1.5% BSA, 0.01% Tween-20, 0.1% NaN₃(ii) a The sample dilution ratio, sample volume to sample buffer volume, was 1: 2.

Example 3 specificity of time-resolved fluorescent microsphere chromatography test strip

As shown in fig. 6, the positive serum of the ovine trichinella against the back, the positive serum of the ovine esophagostomum, and the positive serum sample of the sheep tail nematode are detected, except the positive serum of the ovine trichinella, under a fluorescence immunoassay analyzer, the serum of other non-ovine trichinella pathogens has no signal detected on a T line, and only one band appears on a C line, which is negative.

Example 4 sensitivity of time-resolved fluorescent microsphere chromatography test paper strip

After the positive serum is diluted by multiple times, the diluted sample is respectively dripped into a sample pad, and the fluorescence intensity of the T line is observed under fluorescence immunochromatography after 15 min. The result shows that T, C lines can be clearly seen when the dilution ratio of the positive serum is 1:1600, and the brightness of the T line is very weak but the brightness of the C line is still obvious when the dilution ratio of the positive serum is 1:3200, thereby proving that the detection result is effective. The results are shown in FIG. 7.

As shown in FIG. 8, the test paper prepared in example 2 is used to detect trichinosis in different periods and shows the results of the detection of serum and negative serum.

Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> Shenyang agriculture university

<120> specific antigen for detecting early trichinosis and application thereof

<130> GG21928942A

<160> 4

<170> SIPOSequenceListing 1.0

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<212> PRT

<213> Artificial Sequence

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Met Ile Arg Arg Leu Phe Gln Tyr Thr Ser Met Thr Phe Ala Trp Ile

1 5 10 15

Leu Leu Phe Leu Ser Ala Ala Ser Pro Ser Leu Gly Glu Phe Glu Cys

20 25 30

Gly Val Pro His Phe Lys Pro Tyr Ile Trp Lys Ser Gly Arg Ile Val

35 40 45

Gly Gly Thr Asp Val Arg Pro His Ser His Pro Trp Gln Ile Gln Leu

50 55 60

Leu Lys Ser Glu Thr Gly Gly Tyr Ser Ser Leu Cys Gly Gly Ser Leu

65 70 75 80

Val His Phe Gly Glu Pro Ser Asn Gly Thr Arg Phe Val Leu Thr Ala

85 90 95

Ala His Cys Ile Thr Thr Ser Asn Met Tyr Pro Arg Thr Ser Arg Phe

100 105 110

Thr Val Val Thr Gly Ala His Asn Ile Lys Met His Glu Lys Glu Lys

115 120 125

Lys Arg Ile Pro Ile Thr Ser Tyr Tyr Val Gln His Trp Asn Pro Val

130 135 140

Met Thr Thr Asn Asp Ile Ala Leu Leu Arg Leu Ala Glu Thr Val Tyr

145 150 155 160

Tyr Asn Lys Tyr Thr Arg Pro Val Cys Leu Pro Glu Pro Asn Glu Glu

165 170 175

Leu Thr Pro Gly Asp Ile Cys Val Val Thr Gly Trp Gly Asp Thr Thr

180 185 190

Glu Asn Gly Thr Thr Ser Asn Thr Leu Lys Gln Val Asp Val Lys Ile

195 200 205

Met Lys Lys Gly Thr Cys Ala Asn Val Arg Ser Glu Val Ile Thr Phe

210 215 220

Cys Ala Gly Ala Met Glu Gly Gly Lys Asp Ser Cys Gln Gly Asp Ser

225 230 235 240

Gly Gly Pro Leu Ile Cys Lys Lys Asn Gly Lys Ser Val Gln Phe Gly

245 250 255

Val Val Ser Tyr Gly Thr Gly Cys Ala Arg Lys Gly Tyr Pro Gly Val

260 265 270

Tyr Ala Lys Val Pro Ser Tyr Val Thr Trp Leu Asn Lys Ala Ala Lys

275 280 285

Glu Leu Glu Asn Ser Pro Glu Gly Thr Val Lys Trp Ala Ser Lys Glu

290 295 300

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

305 310 315 320

Thr Gly Ser Arg Pro Thr Ser Pro Ser Ser Gly Ser Arg Pro Thr Tyr

325 330 335

Pro Ser Ser Gly Ser Arg Pro Thr Ser Pro Ser Ser Gly Ser Arg Pro

340 345 350

Thr Tyr Pro Ser Ser Asp Gln Asp Gln His Leu His Leu Val Asp Gln

355 360 365

Asp Pro His Ile His Leu Val Asp Gln Asp Gln His Ile His Ile Leu

370 375 380

Asp Gln Asp Leu Leu Leu Lys Ser Gln Tyr Phe His His Thr Lys Asn

385 390 395 400

Ile Arg Gln Gln Phe Lys Asn Thr Leu Ile Val Tyr Gln Ala Glu Arg

405 410 415

Lys Glu Arg Ser Asn Thr Gln Ser His Arg Met Glu Leu Leu Gln Gln

420 425 430

His Ile Ile Thr Phe Leu Ser Lys Asn Ile Met Ile Asn Ser Leu Leu

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<213> Artificial Sequence

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gatacgactg aaaatggaac tacttctaat actttgaagc aagttgatgt caaaattatg 540

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gagggtggta aagacagttg tcaaggtgat tctggtggcc cactgatatg caagaaaaat 660

gggaaaagtg ttcaattcgg tgtcgttagt tatggtactg gatgcgccag aaaaggttat 720

cccggagtgt atgccaaagt tccatcatat gtcacatggt taaataaagc tgcaaaagaa 780

cttgaaaatt ctcctgaagg aactgtaaaa tgggcttcaa aagaagattc gccagtcgat 840

ttatctacta catcaagacc aactaaccca tatactgggt caagaccgac atctccatct 900

agtggatcaa gacccacata tccatctagt ggatcaagac caacatctcc atctagtgga 960

tcaagaccca catatccatc tagtgatcaa gaccaacatc tccatctagt ggatcaagac 1020

ccacatatcc atctagtgga tcaagaccaa catatccata tactggatca agacctactc 1080

ctcaaaagcc agtatttcca tcataccaaa aatatccgcc agcagttcaa aaatacattg 1140

atagtttacc aagcggaacg caaggaacgc tcgaatacac agtcacacag aatggagtta 1200

ctacaacaac atattatcac ttttctaagt aaaaatatta tgattaattc actactgctc 1260

tga 1263

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<213> Artificial Sequence

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Met Met Leu Thr Ile Ile Thr Val Ile Leu Ile Ser Leu Gly Ser Ser

1 5 10 15

Trp Ala Gln Gly Val Ala Thr Cys Lys Ala Asp Asp Asn Thr Asp Leu

20 25 30

Asn Trp Tyr Phe Val Tyr Lys Pro Pro Asn Ala Leu Gln Thr Lys Ile

35 40 45

Met Gln Ser Gly Gln Asn Pro Ala Trp Ala Arg Ser Ala Gln Ser Ile

50 55 60

Glu Ser Asn Asn Gly His Ser Ile Val Arg Thr Met Ala His Phe Val

65 70 75 80

Ala Glu Asn Gln Asn Ile Lys Val Leu Ala Tyr Ser Asp Asp Pro Pro

85 90 95

Asn Leu Pro Pro Arg Asn Glu Lys Ser Lys Ala Lys Gly Val Leu Leu

100 105 110

Ile Asp Asn Ser Gly Ala Asn Ala Ala Ala Trp Phe Val His Thr Val

115 120 125

Pro Lys Phe Leu Ser His Leu Gly Gly Tyr Ser Trp Pro Gln Thr Glu

130 135 140

Thr Ala Lys Gly His Ile Phe Leu Cys Leu Ser Ile Asn Glu Glu Ser

145 150 155 160

Leu Asn Ala Val Ala Lys Ala Ile Arg Tyr Gln Glu Pro Tyr Ile Tyr

165 170 175

Ala Ser Asn Leu Pro Pro Glu Leu Leu Asn Gln His Asn Glu Leu Ser

180 185 190

Asn Leu Ala Thr Gly Val Glu Ile Arg Ile Thr Pro Phe Leu Glu His

195 200 205

Thr Lys Leu Thr Thr Arg Asn Asn Gly Met Asn Val Gly Ala Phe Gly

210 215 220

Lys His Thr Lys Ser Tyr Ala Asp Met Tyr Glu Arg Val Leu Arg Lys

225 230 235 240

Lys Leu Ser Ala Arg Ile Lys Ile Trp Ala Pro Ser Asp Val Arg Ser

245 250 255

Lys Ser Ile Cys Lys Gly Gln Tyr His Leu Arg Lys Ile Ala Ser Pro

260 265 270

Ile Gln Leu Asp Gly Asp Gln Val His Arg Glu Ala Asp Ser Ala Lys

275 280 285

Trp Ala Leu Val Glu Gly Lys Asn Thr Val Cys Leu Thr Thr Asn Asp

290 295 300

Tyr Lys Thr Thr Glu Lys Arg Ile Pro Gly Ala Ala Val Cys Val Glu

305 310 315 320

Asn Ala Asn Val Tyr Asn Ala Phe Asn Thr Ala Ala Val Asn Val Ala

325 330 335

Ala Cys Asn Met

340

<210> 4

<211> 969

<212> DNA

<213> Artificial Sequence

<400> 4

caaggtgttg caacttgcaa ggcagatgac aatactgatc tcaactggta ttttgtatac 60

aaacctccaa atgctttaca aacaaaaatt atgcagtcgg gacaaaatcc agcttgggca 120

cgttctgcac agtctatcga gagcaacaac ggccattcaa tagttcgaac aatggcacat 180

tttgtagcag agaaccaaaa catcaaagtt cttgcatata gtgacgatcc accaaatttg 240

ccaccaagaa atgaaaaaag caaagccaaa ggagtacttt taattgataa ttcaggagct 300

aatgcagctg cgtggtttgt gcacacagtg cccaaatttt tatcacatct tggtggttat 360

tcttggccac aaacagaaac agcaaaagga cacatatttt tatgtttatc aatcaatgaa 420

gaatctttaa atgctgtagc taaagcaatt cgataccaag aaccatacat atatgcgagt 480

aatttacctc ctgaactttt gaatcaacat aatgaacttt cgaatttggc gacaggagtt 540

gagattcgca taacaccttt tctggagcat acaaaattaa caacaagaaa taatggcatg 600

aatgttggag cttttggaaa acacacaaaa tcatatgcag atatgtatga aagagttctg 660

agaaaaaaac tttctgcaag aatcaaaata tgggcacctt ctgatgtaag atcgaagtca 720

atttgtaagg gacaatacca tcttcgaaaa attgcttctc caatacagct tgatggtgat 780

caagtgcatc gtgaagctga cagtgcaaaa tgggcattag tggaaggaaa gaacacagta 840

tgtcttacaa caaatgatta taagactact gaaaaacgga ttccgggagc tgctgtttgt 900

gttgaaaatg ctaatgttta taacgctttc aatacagcag cagttaatgt tgcagcatgt 960

aatatgtaa 969