CN1730656A - A DNA encoding the N-terminal lipoprotein of Mycoplasma filamentous subspecies - Google Patents

Abstract

The invention discloses a novel DNA encoding N-terminal lipoprotein of mycoplasma filamentous subspecies, its preparation method, its encoded protein and its application in serological detection of bovine infectious pleuropneumonia. The present invention utilizes the in vitro mutagenesis technique to mutate one UGA nucleotide of the 582 nucleotides at the N-terminal of the reading frame of the mycoplasma lipoprotein LppQ gene into UGG, and transforms the mutated lipoprotein LppQ DNA sequence after being connected to an expression vector The host cell is expressed in vitro, and the expressed recombinant protein can be used as an antigen in the serological diagnosis of bovine infectious pleuropneumonia.

Description

A DNA encoding the N-terminal lipoprotein of Mycoplasma filamentous subspecies

technical field

The invention relates to a DNA, in particular to a DNA encoding the N-terminal lipoprotein of mycoplasma filamentous subspecies, its preparation method, its encoded protein and its application in medicine.

Background technique

Mycoplasma mycoide subsp. mycoides SC (MmmSC) is the pathogen that causes bovine contagious pleuropneumonia (Contagious Bovine Pleuroneumonia, CBPP). It is characterized by serous-exudative fibrinous inflammation and serous fibrinous pleuropneumonia, and is listed as a class A infectious disease by the World Organization for Animal Health (OIE). The disease is serious, not only because of the higher morbidity and mortality, but also because it severely limits the foreign trade of cattle and beef products. Therefore, countries where the disease is endemic attach great importance to its control and elimination. CBPP often manifests as subacute or asymptomatic infection, which brings great difficulties to the eradication of the disease. In countries without bovine pneumonia, serological surveillance and slaughterhouse meat surveillance have become important means to control the outbreak and prevalence of the disease.

Isolation of the SC biotype of Mycoplasma filoformis from infected animals is essential for the definitive diagnosis of CBPP. The nutritional requirements of MmmSC are very complex and depend on a variety of nutritional substrates, including vitamins, nucleotides, lipid compounds, fatty acids and amino acids. Propagation of Mycoplasma filamentous subspecies in liquid media requires the inclusion of large amounts of serum as a source of sugar. Aeration culture can increase the culture speed of mycoplasma filamentous subspecies. Mycoplasma mycoplasma filamentous subspecies grows in a filamentous form in the relatively fast-growing medium, and at the end of the culture, it is mainly beaded and short filamentous, and finally only a small amount of small balls.

Several serological methods have been described for more than half a century, including slide agglutination test (SAT), complement fixation test (CFT), agar diffusion test (AGP), passive hemagglutination inhibition test (PHA) and Microagglutination test (MA). Limitations of these trials have also been demonstrated. Although enzyme-linked immunoassay has good sensitivity and is more convenient to operate, the CFT method is still recommended to continue to be used in Europe and Africa. The PHA method appears to be practical, with a 20% higher sensitivity than CFT at the screening test, but approximately 2% false positives in areas where CBPP has never occurred.

Onoviran and Taylor-Robinson first reported the enzyme-linked immunoassay (ELISA) of CBPP in 1979. Poumarat (1989) used ELISA, CFT, PHA and other methods to monitor and compare the antibody responses of bovinegroup7, M.capricolum subsp.capricolum, M.mmLC and M.mycoides subsp capri infected cattle. The ELISA antigen made with M.mmSC strain is more specific than CFT or PHA, and the antibody titer detected in bovine group7 and M.mmLC infected cattle in a specific period is higher. Le Goff (1989, 1990) applied ELISA and CFT to M.mmSC infection and control 5 cattle were tested. Mycoplasma can be isolated from tracheal washes 1-2 weeks before seroconversion. Complement-fixation antibodies appear several weeks earlier than ELISA antibodies, but unlike CFT, ELISA antibodies are positive both during and after excretion of the pathogen.

Wu Yuxiang et al. (1988) reported a microagglutination test and compared it with CFT, proving that MA is more sensitive, specific, easy to operate, and suitable for monitoring a large number of samples.

The final confirmation of mycoplasma infection usually relies on growth inhibition test (GI) or immunofluorescence test (IF). These tests were able to distinguish between the 2 mycoplasma subspecies, but not the LC and SC biotypes. Poumarat (1991) established a rapid dot immunoassay, the main advantage of which is that it is fast and suitable for the detection of a large number of samples.

So far, complement fixation test (CFT) is still the most widely used serological diagnostic method recommended by OIE. The method is more sensitive during the acute phase of CBPP epidemic, and its sensitivity is greatly reduced after the acute phase or after 3 months of infection.

Le Goff and Thiaucourt screened a specific monoclonal antibody from 133 strains of mycoplasma, which could recognize a MmmSC epitope with a molecular weight of about 80KDa and established a competitive ELISA (C-ELISA) for the serological diagnosis of CBPP. )method. We introduced two diagnostic reagents, CFT and C-ELISA, and found that the C-ELISA method was negative for CFT standard positive sera, and the antigenicity of the about 80KDa protein recognized by it has not yet been determined. Therefore, the application of C-ELISA diagnostic method needs to be confirmed by a large number of experiments.

The antigenicity of the lipoprotein LppQ has been established and is widely believed to be present in MmmSC African, European and vaccine strains, but not in other members of the Mycoplasma mycoides cluster. It is reported that the lipoprotein encoded by the N-terminal of the LppQ gene can induce a strong early immune response in naturally infected and experimentally infected cattle, and the antibody lasts for a long time, so it is an ideal diagnostic antigen.

However, by sequencing the entire gene of lipoprotein LppQ, it was found that UGA does not represent a stop codon in the protein translation system of Mycoplasma, but represents tryptophan, and the frequency of use is much higher than that of UGG. In the in vitro expression, the target gene must be mutagenized in vitro to transform UGA into UGG, so that it can be expressed in vitro.

The MmmSC lipoprotein gene has a full length of 1764bp, a reading frame of 1335bp, encoding 445 amino acids, and a molecular weight of about 52.08Kda. After analyzing the antigenicity of the protein, the results show that the N-terminus has good antigenic characteristics, and can induce strong early immune responses in naturally infected and experimentally infected cattle, and the antibody lasts for a long time, so it is an ideal protein. diagnostic antigen. However, there are 10 UGA codons in the protein reading frame, and these codons must be transformed into synonymous codons UGG to be expressed in vitro.

Joachim Frey et al. modified the 1335 nucleotides of the reading frame of lipoprotein LppQ according to its antigenicity, and mutated 9 of them from UGA to UGG, and the molecular weight of the expressed protein was about 48kDa. However, the manipulation of nine nucleotides of the lipoprotein LppQ gene for mutagenesis in vitro is extremely complicated, and the success rate is very low.

Contents of the invention

The technical problem to be solved by the present invention is to provide a new DNA encoding the N-terminal lipoprotein of mycoplasma filamentous subspecies, and the encoded protein can be used as an antigen in the serological diagnosis of bovine infectious pleuropneumonia.

The technical problem to be solved in the present invention is achieved through the following approaches:

Using in vitro mutagenesis technology, one UGA nucleotide of the 582 nucleotides at the N-terminal of the lipoprotein LppQ gene reading frame was mutated into UGG. The nucleotide sequence after the mutagenesis is shown in the sequence table SEQ ID No.1. , the lipoprotein LppQ DNA sequence after mutagenesis is connected with the expression vector and transformed into host cells for in vitro expression, and the expressed recombinant protein (see its amino acid sequence as described in SEQ ID No.2 in the sequence table) is collected and purified and used as an antigen Serological diagnosis of bovine contagious pleuropneumonia.

Detailed description of the invention:

First, extract the genomic DNA of Mycoplasma, design a pair of specific primers containing restriction sites, use the extracted DNA as a template for PCR amplification, recover the amplified specific fragments, and connect the recovered fragments to PUC after enzyme digestion On the carrier, using the carrier as a template, add a pair of carrier universal primers and a pair of mutation primers to carry out polymerase chain reaction, and connect the re-amplified specific fragments to the sequencing carrier for nucleotide sequence determination. After the measurement, it was confirmed that the amplified specific fragment was the desired mutant fragment, and the mutated target fragment was directional cloned into the Pet32a vector, and expressed under the induction of IPTG. SDS-PAGE showed that the molecular weight of the expressed protein was 43Kda. The protein was purified by analysis technology, and the purified protein was used as a coated antigen to detect bovine infectious pleuropneumonia by indirect ELISA method. The results showed that the antigen had very good specificity and sensitivity.

The current serological diagnosis method of bovine contagious pleuropneumonia - the complement fixation test is very cumbersome and requires professional training. In addition, this method still has the disadvantage of low specificity. Recombinant N-terminal lipoproteins have very good specificity, but the technical difficulty of expressing lipoproteins in vitro is difficult and difficult to obtain. This invention uses in vitro site-directed mutagenesis technology to solve the technical problem of lipoprotein expression in vitro. Compared with foreign N-terminal lipoprotein expression, this invention adopts a simpler method to obtain the same effect, and can completely replace foreign products , the N-terminal of the recombinantly expressed lipoprotein can be used as an antigen for serological diagnosis, and the expressed protein is used as a coating antigen to detect bovine infectious pleuropneumonia by indirect ELISA, and the results show that the antigen has very good specificity and sensitivity , the indirect ELISA method established with this antigen is very simple to operate, easy to popularize and apply, and suitable for large-scale serological investigations. Compared with foreign technologies, the operation is simpler, and the specificity and sensitivity meet the requirements.

Description of drawings

Figure 1 The results of the first PCR amplification

Fig. 2 Identification results of recombinant plasmid digestion of the first PCR amplification product

Figure 3 SDS-PAGE electrophoresis results of recombinant expressed protein

Figure 4 Western blotting results of recombinant expressed protein

Detailed ways

The present invention is further described by the following examples. It should be understood that these examples are only for the purpose of illustration, and in no way limit the scope of the present invention.

[Example 1] Preparation of mutant DNA encoding Mycoplasma filamentous subspecies N-terminal lipoprotein

First extract the genomic DNA of Mycoplasma (purchased from the China Veterinary Drug Control Institute), design a pair of specific primers containing the enzyme cutting site EcoR I/Sal I, and the primer sequences are respectively A: 5, GCGGAATTCCCATTAGTTGTTGTTTCATGTA, 3 (SEQ ID No.3 ) and D: 5, GACGTCGACATTAAAGTTTCTAGCTTGTTG, 3 (SEQ ID No.4), using the extracted mycoplasma genomic DNA as a template, utilizing Pyrobest ^TM DNA polymerase and primers A and D to amplify the lipoprotein LppQ N-terminal gene, 100 μl reaction system as follows:

10xBuffer 10.0μl

2.5mMdNTPs 8.0μl

10μmol Primer A 5.0μl

10μmol Primer D 5.0μl

Template DNA 1.0μl

10u/μl Taqpolymerase 1.0μl

Pure water 70μl

Amplification conditions were: pre-denaturation at 95°C for 5 min, denaturation at 94°C for 60 s, annealing at 57°C for 45 s, extension at 72°C for 60 s, and after 30 cycles, extension at 72°C for 10 min. A specific amplified fragment of 582bp was obtained, as shown in Figure 1, where the 1st and 2nd lanes are amplified products, and the 3rd lane is DL2000Marker; the amplified product was digested with EcoR I/Sal I, see Figure 2, where lane 1 is DL2000Marker, lane 2 is the result of EcoR I/Sal I digestion, lane 3 is the result of EcoR I digestion, and lane 4 is the result of Sal I digestion. The digested amplified fragments were ligated to the PUC vector. A pair of mutant primers were designed with primer sequences: 5, TCTTGTTTTTGCCACTCATTTTTTG, 3 (SEQ ID No.5) and: 5, CAAAAAATGAGTGGCAAAAACAAGA, 3 (SEQ ID No. 6). Using the recombinant PUC vector as a template, add a pair of vector universal primers and the mutation primer for PCR amplification. The 100 μl reaction system is as follows:

10xBuffer 10.0μl

2.5mMdNTPs 8.0μl

10μmol Universal Primer 5.0μl

10μmol Primer B 5.0μl

10μmol Primer C 5.0μl

Template DNA 1.0μl

10u/μl Taqpolymerase 1.0μl

Pure water 65μl

The amplification conditions were pre-denaturation at 95°C for 5 minutes, denaturation at 94°C for 60 seconds, annealing at 57°C for 45 seconds, extension at 72°C for 60 seconds, and after 30 cycles, extension at 72°C for 10 minutes. For sequence determination, its nucleotide sequence is described in SEQ ID No.1 in the sequence listing, and the amino acid sequence of its encoded protein is described in SEQ ID No.2 in the sequence listing.

[Example 2] Expression of the mutant DNA encoding the N-terminal lipoprotein of mycoplasma mycoplasma mycoides

The mutated LppQ N-terminal gene and Pet32a vector (purchased from Navagen) were digested with EcoRI/Sal I, the digested fragments were recovered from the gel, ligated with _T4 DNA ligase overnight at 16°C, and the ligated product was transformed into BL21(DE3) Competent cells, the transformed bacteria were spread on LB bacterial medium plate containing 50 μg/ml ampicillin sodium. White colonies were picked, inoculated in LB (containing 50 μg/ml ampicillin sodium) liquid medium, cultured on a shaker at 37° C. for 16 hours, and plasmid DNA was extracted. Use PCR and restriction endonucleases to identify recombinant plasmids, and inoculate LB (containing 50 μg/ml ampicillin sodium) liquid medium at 1% for positive ones, culture on a shaker at 37°C for 3 hours (OD value 0.6-0.8), add IPTG was induced to a final concentration of 1 mM for 3 h. The culture solution was centrifuged at 16,000g for 10min to collect the bacteria, and the collected crude protein was loaded for SDS-PAGE electrophoresis. The protein SDS-PAGE electrophoresis patterns and Western blotting results were shown in Figure 3 and Figure 4, respectively, and finally protein purification by Novagen The kit is obtained by purifying the obtained protein.

[Test Example 1] Serological detection by complement fixation test

1. Material preparation

1. The barbiturate buffer solution used in the test shall be diluted 1:5 when used, and the preparation method is as follows:

Barbiturate buffer (VB) preparation

Sodium Chloride 85g

Barbituric acid 5.75 grams

Sodium Barbital 3.75g

Methyl chloride (MgCl ₂ 6H ₂ O) 1.68g

Calcium chloride (CaCl ₂ 2H ₂ O) 0.37g

Sterilized double distilled water 2000ml

Adjust the pH to 7.3 with hydrochloric acid. Dilute 1:5 with sterile double distilled water.

2. The sheep erythrocyte suspension uses Alfred's solution, and the preparation method is as follows:

                                                                

A solution

Glucose 20.5 grams

Sterilized double distilled water 200ml

Liquid B

Sodium citrate 12.0 g

Sodium chloride 4.2 grams

Sterilized double distilled water 800ml

 Use 5% citric acid to adjust the pH of liquid B to 6.1. Mix liquid A and liquid B, and filter and sterilize with a Chua filter.

3. The preparation method of 6% sheep red blood cell suspension is as follows:

Aseptically collect venous blood from healthy rams, suspend it with Einstein's solution after defibration, wash 3 times with 1500 g, 10 minutes each time. Collect the erythrocyte precipitate, make a 6% suspension with Einstein's solution, store it at 40°C for 48 hours, and use it for no more than 2 weeks at most.

4. Test sample: The antigen used is the international standard bovine infectious pleuropneumonia complement fixation test antigen (purchased from a general biological reagent company), and the positive serum and negative serum are donated by the Portuguese National Veterinary Reference Laboratory (also available from a common biological reagent company). purchased by the company, the effect is equivalent), and it is an international standard product. Hemolysin and complement were purchased from China Veterinary Drug Administration and used according to the instructions. The hemolysin titer was titrated as follows:

Determination of hemolysin potency: 0.1ml of hemolysin was continuously diluted 10 times to 1:1000 as the base solution, and the dilution method is shown in Table 1.

Table 1 Hemolysin dilution method Unit ml Test tube number 1 2 3 4 5 6 7 8 Dilution 1:2000 1:3000 1:4000 1:5000 1:6000 1:8000 1:10000 1:15000 1:1000 hemolysin 1 1 1 1 1 1 1 1 VB 1.0 2.0 3.0 4.0 5.0 7.0 9.0 14.0 Total 2.0 3.0 4.0 5.0 6.0 8.0 10. 15.0

Add various test components according to the procedure in Table 1, in a water bath at 37-38° C. for 30 minutes, and centrifuge at 1500 g for 10 minutes. Mix 100% hemolysis liquid with equal volume of VB to make 50% hemolysis colorimetric tube. The maximum dilution of hemolysin capable of causing 50% hemolysis (HD50) of erythrocyte fluid was used as the titer of hemolysin, and 12 units of HD50 were used.

If the hemolysis degree of the 8th tube in Table 2 reaches 50%, and the control tube has no hemolysis phenomenon, then the potency of this batch of hemolysin is 1:8000, and if 12 units of HD50 are used, the hemolysin should be 8000/ 12 = 666.6-fold dilution.

Table 2 Determination of hemolysin titer Unit ml Test tube number 1 2 3 4 5 6 7 8 9 10 11 hemolysin dilution 1:100 1:1000 1:2000 1:3000 1:4000 1:5000 1:6000 1:8000 1:10000 1:15000 Hemolysin dosage 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 1:20 complement 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 - 6% red blood cells 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 VB 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.8 37-38°C water bath for 30 minutes after shaking Result judgment - - - - - - ± ± ± ± +

Explanation of symbols: "+" inhibits hemolysis, "±" partially hemolysis, "-" total hemolysis.

5. Preparation of sensitized sheep erythrocytes: use hemolysin of 12HD50 (50% hemolysis degree) to mix with equal volume of 6% sheep erythrocytes, bathe in 37° C. water for 30 minutes, and shake once every 5 minutes during the period.

6. The complement was purchased from Harbin Veterinary Biopharmaceutical Factory, and the titer titration method was as follows:

Use VB to dilute the complement, the specific operation is shown in Table 3 and Table 4.

table 3 pipe number 1 2 3 4 5 6 VB(ml) 1.45 1.70 1.95 2.20 2.45 2.70 C'(ml) 0.05 0.05 0.05 0.05 0.05 0.05 Dilution 1:30 1:35 1:40 1:45 1:50 1:55

Table 4 pipe number 1 2 3 4 5 6 complement dilution 1:30 1:35 1:40 1:45 1:50 1:55 VB(ml) 0.5 0.5 0.5 0.5 0.5 0.5 C'(ml) 0.5 0.5 0.5 0.5 0.5 0.5 final complement dilution 1:60 1:70 1:80 1:90 1:100 1:110

Add 25 μl of each dilution of complement from 1:30 to 1:110 into a 96-well micro-reaction plate, add 25 μl of antigen and 25 μl of VB into each complement dilution well, shake and mix well, and then bathe in water at 37°C for 30 minutes. Add 25 μl of sensitized sheep erythrocytes, shake and mix well, bathe in water at 37°C for 30 minutes, and then centrifuge at 125g for 2 minutes to judge the result.

Determination of complement titer: the highest dilution of complement that can dissolve sheep erythrocytes 100% is the complement titer. 2.5U complement was used for detection. For example, complement can dissolve sheep erythrocytes 100% at 1:60, then the complement titer is 60, and it should be diluted 60/2.5=24 times when used.

7. Antigen titer titration method is as follows:

1) Serially dilute the antigen 2 times with VB, from 1:10 to 1:640;

2) Serially dilute the positive serum with VB 2 times, from 1:10 to 1:1280;

3), using a 96-well micro-reaction plate to carry out square array titration of the antigen. The specific operation is shown in Table 5. Add 25 μl of corresponding dilutions of antigen, 25 μl of positive serum, and 25 μl of 2.5 U complement to each well, shake and mix well, and bathe in water at 37°C for 30 minutes. Add 25 μl of sensitized sheep red blood cells to each well, shake and mix well, and then bathe in water at 37°C for 30 minutes. Centrifuge at 125g for 2 minutes to judge the result. At the same time, set 0.5U, 1U, 2.5U complement controls, sensitized red blood cell controls, and anti-complement controls for each antigen dilution.

table 5 1 2 3 4 5 6 7 8 antigen A ++++ ++++ ++++ ++++ +++ + - - 1:10 B ++++ ++++ ++++ ++++ ++++ ++ + - 1:20 C ++++ ++++ ++++ ++++ ++++ +++ + - 1:40 D. ++++ ++++ ++++ ++++ ++++ +++ + - 1:80 E. + ++ +++ ++ ++ + - - 1:160 f - - - - - - - - 1:320 G - - - - - - - - 1:640 positive serum 1:10 1:20 1:40 1:80 1:160 1:320 1:640 1:1280

When all the controls are established, the highest dilution of antigen corresponding to the highest dilution of positive serum with 100% complement bound is the antigen titer. As shown in Table 5, when the positive serum ratio is 1:160 and the antigen ratio is 1:80, the antigen-antibody complex can bind 100% complement, then the antigen titer is 1:80. 2 units are used for detection, and the antigen is diluted 80/2=40 times.

Note: All the biochemical reagents used above can be purchased from biochemical reagent companies.

2. Test method

1. The tested serum, standard negative and positive serum were diluted 1:10 with barbiturate buffer and inactivated in a 56°C water bath for 30 minutes.

2. Add 25 μl of antigen, 25 μl of serum to be tested, and 25 μl of 2.5 U complement in each well of a 96-well micro-reaction plate, shake and mix well, and bathe in water at 37°C for 30 minutes.

3. Add 25 μl of sensitized sheep red blood cells to each well, shake and mix well, and then bathe in water at 37°C for 30 minutes.

4. Set up standard positive serum, negative serum, complement control, hemolysin control, and antigen anti-complement activity control.

3. Judgment

1. Centrifuge the 96-well microplate at 125g for 2 minutes or stand still for 10 minutes. When the negative control, positive control, complement control, and red blood cell control are established, determine the percentage of complement binding in the tested well.

2. The method for determining the percentage of complement fixation is as follows:

Table 6 Judgment of complement fixation percentage percent complement fixation judgement result 100% ++++ 75% +++ 50% ++ 25% + 0% -

3. Judgment criteria:

When the serum is diluted 1:10, ++++ is positive;

When the serum is diluted 1:10, +, ++, +++ are suspected;

When the serum was diluted 1:10, - was negative.

For suspected samples, re-collect serum for re-examination, and if it is still suspected, conduct pathological and etiological examinations.

4. Test results

The test results are shown in Table 7.

Table 7 The detection results of positive bovine serum of artificially infected bovine contagious pleuropneumonia by complement fixation test (CFT) Cow number only Blood collection time after artificial infection (days) Test sample positive control negative control complement control RBC control 1 twenty one Positive Positive Negative set up set up 1 28 Positive Positive Negative set up set up 3 9 Positive Positive Negative set up set up 3 14 Positive Positive Negative set up set up 3 43 Positive Positive Negative set up set up 4 9 Positive Positive Negative set up set up 4 14 Positive Positive Negative set up set up 4 43 Positive Positive Negative set up set up 4 58 Positive Positive Negative set up set up 5 9 Positive Positive Negative set up set up 5 14 Positive Positive Negative set up set up 5 twenty one Positive Positive Negative set up set up 5 28 Positive Positive Negative set up set up 7 9 Positive Positive Negative set up set up 7 14 Positive Positive Negative set up set up 7 twenty one Positive Positive Negative set up set up 8 9 Positive Positive Negative set up set up 8 14 Positive Positive Negative set up set up 8 twenty one Positive Positive Negative set up set up 8 28 Positive Positive Negative set up set up 9 9 Positive Positive Negative set up set up 9 14 Positive Positive Negative set up set up 9 28 Positive Positive Negative set up set up

The results show that the recombinant expressed protein of the present invention is used as an antigen to detect bovine contagious pleuropneumonia by using the complement fixation test method, and the accuracy of the detection result is high, indicating that the antigen has high specificity and sensitivity, and can be used for bovine contagious pleuropneumonia serological diagnosis.

[Test Example 2] Serological detection of bovine infectious pleuropneumonia using recombinantly expressed N-terminal lipoprotein of mutant Mycoplasma filamentous subspecies

1. Test materials:

1. Test sample: use the purified protein of the embodiment of the present invention as the coating antigen

2. The positive control and the negative control were donated by the Portuguese National Veterinary Reference Laboratory (it can also be purchased from a general biological reagent company, the effect is equivalent), and they are international standard products. The blank control was 100 μl substrate solution plus 50 μl stop solution.

3. Reagents: fish gelatin, Tween 20, OPD (o-phenylenediamine) (all can be purchased from common biological reagent companies).

2. Test method

The protein purified in Example 2 was coated with a 96-well flat-bottomed polystyrene microtiter plate at 1 μg/ml, and the specific test procedure was as follows:

1. Coating antigen: Dilute the antigen to the working concentration with pH 9.6, 0.05M carbonate buffer, add 100 μl to each well with a microsampler, cover with parafilm, and coat overnight at 4°C . The next day, pour off the coating solution and wash with Stat Fax 2600 automatic plate washer.

2. Sealing: Add 100 μl 3% fish gelatin to each well, incubate in a humid box at 37°C for 90 minutes, shake dry and wash as above.

3. Adding the tested serum: the serum was diluted 1:80 times with PBS (PBST) containing 0.05% Tween 20, and 100 μl was added to each well. Place in a humid box and incubate at 37°C for 30 minutes, shake dry and wash as above.

4. Add enzyme conjugates: Dilute the enzyme conjugates 1:2000 times with PBST (phosphate buffered saline), add 100 μl to each well, incubate at 37°C for 1 hour in a wet box, spin dry and wash as above.

5. Add substrate solution: add 100 μl of OPD (o-phenylenediamine) solution to each well, and place in a dark box at room temperature for 30 minutes.

6. Add stop solution: add 50 μl of 2M H ₂ SO ₄ to each well to stop the reaction.

7. Determination of optical density (OD): After the reaction is terminated, use a Stat Fax 2600 automatic microplate reader to measure the OD value of each well at 492nm.

Standard positive, standard negative and substrate stop solution blank control wells were set up in each test. The OD value of standard positive serum samples is between 0.8-1.0; the OD value of standard negative serum samples is between 0.20-0.25.

3. Test results

The test results are shown in Table 8.

Table 8 Application of indirect ELISA test to the detection results of artificially infected bovine contagious pleuropneumonia positive bovine serum Cow number only Blood collection time after artificial infection (days) positive control negative control Substrate stop solution blank control Test sample 1 twenty one 0.97 0.23 0.08 0.779 1 28 0.97 0.23 0.08 0.682 3 9 0.97 0.23 0.08 0.304 3 14 0.97 0.23 0.08 0.622 3 43 0.97 0.23 0.08 0.233 4 9 0.97 0.23 0.08 1.732 4 14 0.97 0.23 0.08 0.919 4 43 0.97 0.23 0.08 0.962 4 58 0.97 0.23 0.08 0.592 5 9 0.97 0.23 0.08 0.375 5 14 0.97 0.23 0.08 0.528 5 twenty one 0.97 0.23 0.08 0.547 5 28 0.97 0.23 0.08 0.682 7 9 0.97 0.23 0.08 0.84 7 14 0.97 0.23 0.08 0.728 7 twenty one 0.97 0.23 0.08 1.78 8 9 0.97 0.23 0.08 0.59 8 14 0.97 0.23 0.08 0.692 8 twenty one 0.97 0.23 0.08 0.802 8 28 0.97 0.23 0.08 0.801 9 9 0.97 0.23 0.08 0.276 9 14 0.97 0.23 0.08 0.311 9 28 0.97 0.23 0.08 0.309

Note: The numerical result of the test sample is greater than or equal to 0.25 is positive

The results show that: the recombinant expressed protein of the present invention is used as an antigen to detect bovine contagious pleuropneumonia by using the indirect ELISA test method, and the detection result accuracy rate is high, indicating that the antigen has very high specificity and sensitivity, and can be used for bovine contagious pleuropneumonia serological diagnosis.

Sequence Listing

<110> Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences

<120> A DNA encoding the N-terminal lipoprotein of Mycoplasma filamentous subspecies

<160>6

<210>1

<211>573

<212>DNA

<213> Artificial sequence

<400>1

ccattagttg ttgtttcatg taaaacttca aattttaata acaataaacc taataataat 60

caacaaaaaa aagagcaagt aagtaaaatt gatatttcaa gttttaaaga taaaattgaa 120

ccaaaaaatg agtggcaaaa acaagatgtt ttacaagcat tactaaaaat aaaagggcta 180

gataaattaa ctcaaaatga ttttaatttt aatattaaga aagctaattt attacgtaat 240

ggacaattga taattaaatc taaagacgat tctaaaatta taaaaggtga actaagttta 300

gaaattaaaa aactaaatag agttaaaaaa gttgaaacaa aatataatga tactagaact 360

gaggttttag taattggtta tgatgaaaat ggaaaaattt ctgggtttgc tcaaactgtt 420

aaaaaagttc ctgaaaaact accagaagaa atttatagtc tagagcgtgc ttttttaaaa 480

aataattctg ataaaattga aaatcttgat aaatgggaca catctaatat agtaagtatg 540

tcttcaatgt ttcaacaagc tagaaacttt aat 573

<210>2

<211>191

<212>PRt

<213> Artificial sequence

<400>2

Pro Leu Val Val Val Ser cys Lys Thr Ser Asn Phe Asn Asn Asn

1 5 10 15

Lys Pro Asn Asn Asn Gln Gln Lys Lys Glu Gln Val Ser Lys Ile

20 25 30

Asp Ile Ser Ser Phe Lys Asp Lys Ile Glu Pro Lys Asn Glu Trp

35 40 45

Gln Lys Gln Asp Val Leu Gln Ala Leu Leu Lys Ile Lys Gly Leu

50 55 60

Asp Lys Leu Thr Gln Asn Asp Phe Asn Phe Asn Ile Lys Lys Ala

65 70 75

Asn Leu Leu Arg Asn Gly Gln leu Ile Ile Lys Ser Lys Asp Asp

80 85 90

Ser Lys Ile Ile Lys Gly Glu Leu Ser Leu Glu Ile Lys Lys Leu

95 100 105

Asn Arg Val Lys Lys Val Glu Thr Lys Thr Asn Asp Thr Arg Thr

110 115 120

Glu Val Leu Val Ile Gly Thy Asp Glu Asn Gly Lys Ile Ser Gly

125 130 135

Phe Ala Gln Thr Val Lys Lys Val Pro Glu Lys Leu Pro Glu Glu

140 145 150

Ile Ile Ser Leu Glu Arg Ala Phe Leu Lys Asn Asn Ser Asp Lys

155 160 165

Ile Glu Asn Leu Asp Lys Trp Asp Thr Ser Asn Ile Val Ser Met

170 175 180

Ser Ser Met Phe Gln Gln Ala Arg Asn Phe Asn

185 190

<210>3

<211>31

<212>DNA

<213> Artificial sequence

<400>3

gcggaattcc cattagttgt tgtttcatgt a 31

<210>4

<211>30

<212>DNA

<213> Artificial sequence

<400>4

gacgtcgaca ttaaagtttc tagcttgttg 30

<210>5

<211>15

<212>DNA

<213> Artificial sequence

<400>5

tcttgttttt gccactcatt ttttg 15

<210>6

<211>15

<212>DNA

<213> Artificial sequence

<400>6

caaaaaatga gtggcaaaaa caaga 15

Claims (10)

1, a kind of DNA of the thread subspecies N end of the thread mycoplasma lipoprotein of encoding is characterized in that: have the described sequence of sequence table SEQ ID No.1.

2, the coded protein of the DNA of claim 1 is characterized in that: have the described sequence of sequence table SEQ IDNo.2.

3, the preparation method of the described DNA of claim 1 may further comprise the steps:

1) genomic dna of extraction mycoplasma designs a pair of Auele Specific Primer, is that template is carried out pcr amplification with the mycoplasma genomic dna that is extracted, the specific fragment that increases after cutting, enzyme is connected on the PUC carrier,

2) designing a pair of mutant primer, is template with this reorganization PUC carrier, adds a pair of carrier universal primer and this mutant primer and carries out pcr amplification, collect amplification to specific fragment promptly.

4, according to the described preparation method of claim 3, wherein the described Auele Specific Primer of step 1) has sequence table SEQ ID No.3 and the described sequence of SEQ ID No.4.

5, according to the described preparation method of claim 3, wherein step 2) described mutant primer has sequence table SEQ ID No.5 and the described sequence of SEQ ID No.6.

6, the expression vector that contains the described DNA of claim 1.

7,, it is characterized in that described expression vector is Pet22b, Pet30a, PGEX-6p-1, pProEXHTa, the Pet32a carrier that contains the described DNA of claim 1 according to the described expression vector of claim 6.

8,, it is characterized in that described expression vector is the Pet32a carrier that contains the described DNA of claim 1 according to the described expression vector of claim 7.

9, the clone that contains the described DNA of claim 1.

10, the described DNA of claim 1 is or/and the application of the described protein of claim 2 in preparation contagious bovine pleuropneumonia serology detection reagent.

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