CN111789941A

CN111789941A - Bivalent inactivated vaccine for mycoplasma pneumonia and chlamydia psittaci disease of goats and preparation method thereof

Info

Publication number: CN111789941A
Application number: CN201910279104.3A
Authority: CN
Inventors: 李敏; 刘少蓉; 高强; 段跃强
Original assignee: Inner Mongolia Huaxi Biotechnology Co ltd
Current assignee: Inner Mongolia Huaxi Biotechnology Co ltd
Priority date: 2019-04-09
Filing date: 2019-04-09
Publication date: 2020-10-20
Anticipated expiration: 2039-04-09
Also published as: CN111789941B

Abstract

The invention discloses a bivalent inactivated vaccine for mycoplasma capricolum pneumonia and chlamydia psittaci disease and a preparation method thereof. The active ingredients of the bivalent inactivated vaccine comprise MOMP proteins of inactivated mycoplasma filiformis goat subspecies, inactivated mycoplasma ovipneumoniae and inactivated chlamydia psittaci. The amino acid sequence of the MOMP protein of Chlamydia psittaci is shown as sequence 2 in the sequence table. The mycoplasma ovipneumoniae is mycoplasma ovipneumoniae SH-01CGMCC No. 16503. The filamentous mycoplasma goat subspecies is filamentous mycoplasma goat subspecies GT-01CGMCC No. 16504. Experiments prove that the bivalent inactivated vaccine has good safety for immunizing both experimental animals and target animals, and can effectively prevent the experimental animals and the target animals from being infected by the goat mycoplasma pneumonia and the chlamydia psittaci. Has important significance for preventing and controlling the prevalence and the spread of the mycoplasma pneumoniae of goats and the chlamydia psittaci disease. The invention has important application value.

Description

Bivalent inactivated vaccine for mycoplasma pneumonia and chlamydia psittaci disease of goats and preparation method thereof

Technical Field

The invention belongs to the field of biological products for livestock, and particularly relates to a combined inactivated vaccine for mycoplasma capricolum pneumonia and chlamydia psittaci disease and a preparation method thereof.

Background

Mycoplasma capricolum pneumonia is a contagious disease that is prevalent in goats and sheep, caused by Mycoplasma filiformis capricolub (Mmc) and/or Mycoplasma ovipneumoniae (Movi) and/or Mycoplasma capricolum (Mcc). The mycoplasma pneumonia of goats is most common in late summer and autumn, and is characterized by the main pathological changes of high fever, cough, dyspnea, progressive emaciation, interstitial proliferative inflammation of lung, liquid reactivity of chest and pleura and cellulose inflammation, and once a flock is infected, the infection rate is high. Mycoplasma pneumonia of goats is currently occurring widely in many countries and regions, including middle asia, north africa, middle east and other areas where sheep are maintained. In China, mycoplasma pneumonia of goats caused by Mmc and Movi is prevalent, and causes serious harm to the sheep raising industry.

In recent years, researchers at home and abroad think that the aetiology of the contagious pleuropneumonia of sheep is complex and various pathogenic bacteria types exist. Mcc, mcm and mycoplasma Capricolum subsp. pneumoniae (Mccp) are pathogens causing contagious pleuropneumonia diseases in goats, which are related to the higher relativity mycoplasma mycoides mycoplamamioides (mm cluster). Movi can cause hyperplastic, interstitial and infectious pleuropneumonia in sheep and goats. Domestic research proves that two pathogenic bacterial types of Mmc and Movi are dominant bacterial types which are widely popular in goat mycoplasma pneumonia diseases in many areas of China. According to the studies of Dungguanming et al (1991), there was no cross-reaction between Mmc and Movi mycoplasma in serology.

Mmc is a tiny, pleomorphic microorganism, without a cell wall, gram-negative. The cells are in various forms such as spherical, rod-like, arc-like, comma-like, pear-like, and most commonly spherical. The size of the thallus is larger, the diameter of the mycoplasma thallus is generally 125-500nm, and the diameter of the Mmc is 150-1250 nm. Mmc formed clear microcolonies on solid media, slightly larger than other Mycoplasma colonies. Under a low power optical microscope, most colonies of Mmc are typically fried egg-shaped, and the center bulge of the colony is in a yellowish-brown nipple shape. Movi also has no cell wall and is gram negative. The thallus is highly polymorphous, such as small sphere, ellipse, filamentous, rod-like, and the like, and is common in small sphere. Movi formed clear, fine, needle-like colonies on solid medium. Under a low power optical microscope, Movi is in a round bulge and mulberry-like shape, has no central extrusion and is not fried into an egg shape. In the natural state, Mmc only infects goats, but generally does not infect sheep; movi can infect sheep and goat, and common lambs are susceptible. In late winter, early spring and late autumn, due to sudden temperature change, the breeding density is higher, and particularly in sheeppens fed in houses, mycoplasma pneumonia of goats is easy to occur and prevail. The disease is endemic, the diseased sheep are the main infection source, the droplet infection of the respiratory tract is the main transmission mode, and the outbreak of the new epidemic area is almost caused by the introduction of the diseased sheep which is not quarantined. The disease has a fast spread speed, and the disease can spread to the whole population within 20 days.

People are working on finding an effective method for preventing and controlling mycoplasma pneumonia of goats. Due to the characteristics of mycoplasma, the pathogens are difficult to be cleared from the sheep body by using the medicine, and the disease can relapse after the medicine is stopped. On the other hand, because the use of antibiotics causes the residual antibiotics in the product, which is harmful to human health, vaccination is the first method for preventing mycoplasmosis. All countries generally develop inactivated vaccines suitable for local areas according to local specific epidemics for immunization. The cross protection is lacked between the mycoplasma filiformis goat subspecies and the mycoplasma ovipneumoniae which are popular in China, the mycoplasma filiformis goat subspecies and the mycoplasma ovipneumoniae are often mixed to be infected, and the same mycoplasma is easy to be mutated to cause antigen difference.

Chlamydophila psittaci (Cps) is an obligate intracellular parasitic zoonotic pathogen belonging to Chlamydophila (Chlamydophila) of Chlamydophilae. Cps have strong infectivity on birds, poultry and livestock. CpS can infect more than 130 kinds of birds, and the infected birds have symptoms of dyspnea, low mood, and the like, and can die seriously. After cattle, sheep and other mammals are infected with Cps, the Cps are clinically characterized by dyspnea, arthritis, conjunctivitis, fever, abortion, death and the like, and huge economic loss is caused to the animal husbandry. Domestic animal chlamydia infection and psittacosis chlamydia infection also exist in poultry. 8% -50% of chlamydial infection generally exists in some large-scale livestock farms which are introduced from abroad frequently, and the abortion rate and the stillbirth rate of infected animal groups in the livestock farms which are infected by the chlamydial are far higher than those in the livestock farms which are not infected by the chlamydial. In recent years, the sheep breeding disorder diseases caused by psittacosis chlamydia are continuously reported, and the sheep flocks of rural sheep farms and households in Helen city are attacked by chlamydia to different degrees, and are clinically characterized by fever, abortion, death, teratogenic lambs and weak lambs, so that serious economic loss is caused to the sheep industry.

With the continuous development and progress of molecular biology technology, genetic engineering subunit vaccines with the characteristics of good safety performance, easy large-scale production and the like are more and more concerned by researchers and users. Research data indicate that the major outer membrane protein of chlamydia (MOMP), also known as outer membrane protein a (ompa), accounts for approximately 60% of the outer membrane and is a species-specific antigen.

Disclosure of Invention

The invention aims to provide a bivalent inactivated vaccine capable of preventing mycoplasma pneumoniae of goats and chlamydia psittaci simultaneously.

The invention firstly protects a bigeminal inactivated vaccine of goat mycoplasma pneumonia and Chlamydia psittaci disease, and the active ingredients of the bigeminal inactivated vaccine can comprise inactivated mycoplasma filiformis goat subspecies, inactivated mycoplasma ovipneumoniae and inactivated MOMP protein of Chlamydia psittaci;

the MOMP protein of chlamydia psittaci may be a1) or a2) or a 3):

a1) the amino acid sequence is protein shown as a sequence 2 in a sequence table;

a2) a fusion protein obtained by connecting labels to the N end or/and the C end of the protein shown in the sequence 2 in the sequence table;

a3) and (b) the protein shown in a1) or a2) is subjected to substitution and/or deletion and/or addition of one or more amino acid residues to obtain the protein with the same function.

The active ingredients of the bivalent inactivated vaccine can be specifically composed of MOMP proteins of inactivated mycoplasma filiformis goat subspecies, inactivated mycoplasma ovipneumoniae and inactivated chlamydia psittaci.

In any of the above-mentioned bivalent inactivated vaccines, the ratio of the MOMP proteins of the inactivated mycoplasma filiformis goat subspecies, the inactivated mycoplasma ovipneumoniae and the inactivated chlamydia psittaci may be 1.2 × 10⁹ccu：(1.2×10⁸-1.2×10¹⁰) ccu (e.g. 1.2X 10)⁸-1.2×10⁹ccu、1.2×10⁹-1.2×10¹⁰ccu、1.2×10⁸ccu、1.2×10⁹ccu or 1.2X 10¹⁰ccu): (1-3) mg (e.g., 1-1.8mg, 1.8-3mg, 1mg, 1.8mg, or 3 mg).

In any of the above-mentioned bivalent inactivated vaccines, the ratio of the MOMP proteins of the inactivated mycoplasma filiformis goat subspecies, the inactivated mycoplasma ovipneumoniae and the inactivated chlamydia psittaci may be specifically 1.2 × 10⁹ccu：1.2×10⁹ccu：1.8mg。

The invention also provides an inactivated vaccine for mycoplasma capricolum pneumonia, and the active ingredients of the inactivated vaccine can comprise inactivated mycoplasma filiformis subspecies capricolum and inactivated mycoplasma ovipneumoniae.

The active ingredients of the inactivated vaccine for mycoplasma capricolum pneumonia can be inactivated mycoplasma filiformis subspecies capricolum and inactivated mycoplasma ovipneumoniae.

Any mycoplasma ovipneumoniae can be specifically mycoplasma ovipneumoniae SH-01, and the preservation number of the mycoplasma ovipneumoniae SH-01 in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No. 16503.

Any one of the mycoplasma filiformis goat subspecies can be specifically mycoplasma filiformis goat subspecies GT-01, and the preservation number of the mycoplasma filiformis goat subspecies in the China general microbiological culture Collection center is CGMCC No. 16504.

The mycoplasma ovipneumoniae SH-01 and the mycoplasma filiform goat subspecies GT-01 are obtained by screening 2684 parts of suspected diseased goat lung tissues and pharynx swabs suffering from mycoplasma pneumonia in sheep farms in inner Mongolia, Chongqing, Anhui, Qinghai, Yunnan, Guizhou and Guangxi areas of China through methods such as separation, serology and genetic identification.

Any of the above vaccines may further comprise an adjuvant.

The invention also provides a preparation method of the inactivated vaccine for mycoplasma capricolum pneumonia, which comprises the following steps:

(a1) respectively inoculating the filamentous mycoplasma goat subspecies and the mycoplasma ovipneumoniae to a culture medium for culturing mycoplasma, and culturing to obtain corresponding virus culture solutions;

(a2) after the step (a1) is finished, respectively taking two virus culture solutions, and inactivating and purifying to obtain corresponding virus stock solutions;

(a3) and (c) after the step (a2) is finished, mixing the two virus stock solutions with an adjuvant to obtain the inactivated vaccine for the mycoplasma pneumoniae of the goats.

The invention also provides a preparation method of the bivalent inactivated vaccine for the mycoplasma capricolum pneumonia and the chlamydia psittaci disease, which comprises the following steps:

(c1) respectively inoculating the filamentous mycoplasma goat subspecies and the mycoplasma ovipneumoniae to a culture medium for culturing mycoplasma, and culturing to obtain corresponding virus culture solutions;

(c2) after the step (c1) is finished, respectively taking two virus culture solutions, and inactivating and purifying to obtain corresponding virus stock solutions;

(c3) preparing MOMP protein of the Chlamydia psittaci;

(c4) after the step (c3) is completed, taking the MOMP protein of the Chlamydia psittaci, and inactivating;

(c5) and (c) mixing the two virus stock solutions obtained in the step (c2), the MOMP protein of the inactivated Chlamydia psittaci obtained in the step (c4) and an adjuvant to obtain the bivalent inactivated vaccine for the goat mycoplasma pneumonia and the Chlamydia psittaci disease.

The invention also protects the mycoplasma filiformis goat subspecies GT-01, the preservation number of which in the China general microbiological culture Collection center of the Committee for preservation management of microorganisms is CGMCC No. 16504.

The present invention also protects S1) or S2).

S1) the use of the MOMP protein of any one of the chlamydia psittaci and/or any one of the mycoplasma filiform goat subspecies GT-01 and/or mycoplasma ovipneumoniae in the preparation of a combined inactivated vaccine for goat mycoplasma pneumonia and chlamydia psittaci disease.

S2) use of any one of the above mentioned mycoplasma filiformis goat subspecies GT-01 in the preparation of inactivated vaccine for mycoplasma capricolum pneumonia.

In the application, the mycoplasma ovipneumoniae can be mycoplasma ovipneumoniae SH-01, and the preservation number of the mycoplasma ovipneumoniae SH-01 in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No. 16503.

The vaccine can be prepared into subcutaneous injection, intramuscular injection or needleless transdermal injection.

Above, the adjuvant may be ISA201, SP01, MF59, or aluminum hydroxide.

In the above, "the mycoplasma filiformis subspecies capricolum and the mycoplasma ovipneumoniae are respectively inoculated to a culture medium for culturing mycoplasma, and cultured to obtain a corresponding virus culture solution", the culture medium for culturing mycoplasma can be specifically a mycoplasma liquid culture medium composed of a basic culture solution, horse serum, a secondary solution and Penicillin. The ratio of the mycoplasma liquid culture medium can be 15mL of basal medium, 4mL of horse serum, 1mL of auxiliary medium and 120 ten thousand units of Penicilin. The temperature of the culture can be 35-39 deg.C (e.g., 35-37 deg.C, 37-39 deg.C, 35 deg.C, 37 deg.C, or 39 deg.C). The culture parameter can be that the tank pressure is kept at 0.05 MPa; the pH value of the aqueous solution of 10% (v/v) ammonia water is automatically adjusted to be kept at 7.8; the initial rotation speed is 200rpm, the dissolved oxygen continuously decreases along with the growth of thalli, and the dissolved oxygen is adjusted by automatically adjusting the rotation speed and the ventilation capacity to keep the dissolved oxygen at 20-30%.

The preparation method of the basic culture solution can be as follows: adding 4500mL of distilled water into PPLO broth126g and 18g of Glucose for full dissolution; then 10ml of 0.4% (m/v) phenol red solution (solvent is 0.1mol/L NaOH aqueous solution) is added dropwise, the pH value is adjusted to 7.6-7.8 by 1mol/L NaOH aqueous solution, and autoclaving is carried out for 15min at 121 ℃.

The preparation method of the auxiliary liquid comprises the following steps: adding 300mL of distilled water into 8g of sodium pyruvate and 25g of yeast extract, and fully dissolving; then, the mixture was filtered through a filter having a pore size of 0.22 μm.

As mentioned above, in the above-mentioned "two virus culture solutions are respectively taken, and inactivated and purified to obtain corresponding virus stock solutions", formaldehyde can be used for inactivation. Can be purified by concentration (100KD) by ultrafiltration. The formaldehyde inactivation method can be as follows: adding formaldehyde solution into virus culture solution according to 0.2% (v/v) of fermentation broth volume, and culturing at 23-27 deg.C (such as 23-25 deg.C, 25-27 deg.C, 23 deg.C, 25 deg.C or 27 deg.C) for 8-12h (such as 8-10h, 10-12h, 8h, 10h or 12 h).

As described above, the "MOMP protein of Chlamydia psittaci, inactivation" may be inactivated with formaldehyde. The formaldehyde inactivation method can be as follows: adding 0.1% (v/v) formaldehyde solution into protein solution of Chlamydia psittaci MOMP protein, and culturing at 23-27 deg.C (such as 23-25 deg.C, 25-27 deg.C, 23 deg.C, 25 deg.C or 27 deg.C) for 23-27h (such as 23-25h, 25-27h, 23h, 25h or 27 h).

The preparation method of the MOMP protein of the Chlamydia psittaci can be as follows:

1. constructing recombinant escherichia coli;

2. fermenting and culturing recombinant escherichia coli (IPTG is added during fermentation to induce) to obtain a zymophyte liquid;

3. taking the zymophyte liquid, centrifuging and collecting thalli;

4. taking the thalli, and carrying out heavy suspension to obtain a heavy suspension;

5. taking the heavy suspension, crushing, centrifuging and collecting a precipitate;

6. adding inclusion body dissolving solution (such as Tris-HCl buffer solution (8M) containing urea and 5mM DTT, pH8.0, and 0.02M) into the precipitate for fully dissolving; then centrifuging and collecting the supernatant (the supernatant is denatured MOMP protein);

7. and (3) taking the supernatant, performing gradient dialysis by using PBS (phosphate buffer solution) buffer solutions with different urea contents (such as 8mol/L, 6mol/L, 4mol/L, 2mol/L and 0mol/L) respectively to perform protein renaturation, collecting renatured protein solution, and filtering to obtain the protein solution.

The recombinant Escherichia coli can be specifically the recombinant plasmid pET-22b-MOMP described in the examples.

The preparation method of the MOMP protein of Chlamydia psittaci is specifically shown in example 4.

Experiments prove that the bivalent inactivated vaccine for the mycoplasma capricolum pneumonia and the chlamydia psittaci prepared by the invention has good safety for immune experimental animals (such as guinea pigs, rabbits and Beagle dogs) and target animals (such as goats), and can effectively prevent the mycoplasma capricolum pneumonia and the chlamydia psittaci from infecting the experimental animals and the target animals. Has important practical significance for preventing and controlling the prevalence and the spread of the mycoplasma pneumoniae of goats and the chlamydia psittaci disease. The invention has important application value.

Drawings

FIG. 1 shows the results of Dienes staining of 5 Mycoplasma filiformis goat subspecies.

FIG. 2 shows the results of Dienes staining of 5 M.ovis pneumoniae.

FIG. 3 shows the result of SDS-PAGE in example 3.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.

The experimental procedures in the following examples are conventional unless otherwise specified.

The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

The quantitative tests in the following examples, all set up three replicates and the results averaged.

PPLO broth is a product of BD corporation. PPLO Agar is a product of Qingdao Haibo Biotech Co. Yeast Extract is a product of OXOID, Inc. in the United kingdom. Penicilin is a product of North China pharmaceutical products, Inc. Glucose, phenol red, NaOH and sodium pyruvate are all products of national pharmaceutical group chemical reagents, Inc. Horse serum is a product of GIBCO company.

Basic culture solution: adding 4500mL of distilled water into PPLO broth126g and 18g of Glucose for full dissolution; then 10mL of 0.4% (m/v) phenol red solution (solvent is 0.1mol/L NaOH aqueous solution), pH value is adjusted to 7.6-7.8 by 1mol/L NaOH aqueous solution, and autoclaving is carried out for 15min at 121 ℃.

Auxiliary liquid: adding 300mL of distilled water into 8g of sodium pyruvate and 25g of yeast extract, and fully dissolving; then, the mixture was filtered through a filter having a pore size of 0.22 μm.

Mycoplasma liquid culture medium: consists of 4500mL of basal medium, 1200mL of horse serum, 300mL of auxiliary solution and 120 ten thousand units of Penicilin.

Mycoplasma solid culture medium: adding 750mL of distilled water into PPLO Agar20g and Glucose3g, fully dissolving, and autoclaving at 121 ℃ for 15 min; when the temperature is cooled to about 55 ℃, 200mL of horse serum and 50mL of auxiliary liquid are added, and 20 ten thousand units of Penicilin are added and mixed evenly; pouring into autoclaved glass plates (about 30mL of each plate), air-drying in a clean bench, and solidifying to obtain mycoplasma solid culture medium.

Chlamydia psittaci CG1 (described in Lepeng, He Jun, Zhu hong, Liyanwei, Yonghui, Dunqing, Song Li Hua, B598_0590 gene of Chlamydia psittaci encodes inclusion body membrane protein. Biotechnology communication 2016 (Mr.) 01) was given as a gift to professor Song Li Hua, academy of military medical sciences.

Example 1 seed batch preparation of Mycoplasma filiformis subspecies goat and Mycoplasma ovipneumoniae

The inventor collects 2684 samples of sheep lung tissues and throat swabs suffering from mycoplasma pneumonia suspected diseases in the sheep farms of Chongqing city, Anhui province, Qinghai province, Yunnan province, Xinjiang Uygur autonomous region, Guizhou province, Guangxi Zhuang autonomous region and inner Mongolia autonomous region, and selects and determines the dominant epidemic strains of the filamentous mycoplasma goat subspecies and the dominant epidemic strains of the sheep mycoplasma pneumonia through separation and verification. The method comprises the following specific steps:

1. preliminary screening

(1) Putting the sample into a culture bottle containing mycoplasma liquid culture medium (throat swab is directly put in, and sheep lung tissue is put in after being ground), and putting the sample into a constant-temperature incubator at 37 ℃ for culture. When the color of the Mycoplasma liquid culture medium changed from red to yellow, it was filtered through a filter having a pore size of 0.45. mu.m, and the filtrate was collected.

(2) After the completion of the step (1), the filtrate was inoculated on a mycoplasma solid medium and cultured in a 37 ℃ incubator for 1 week.

2. Double sieve

After the step 1 is completed, selecting and purifying colonies from a mycoplasma solid culture medium for three times to obtain the mycoplasma pneumoniae strain. The procedure for each colony purification was as follows:

(1) selecting bacterial colony from mycoplasma solid culture medium, inoculating to mycoplasma liquid culture medium, and culturing in 37 deg.C constant temperature incubator. When the color of the mycoplasma broth changes from red to yellow (caused by mycoplasma growth), it is filtered through a filter with a pore size of 0.45 μm, and the filtrate is collected.

(2) After the step (1) is completed, inoculating the filtrate onto a mycoplasma solid culture medium, and culturing in a constant-temperature incubator at 37 ℃.

After primary screening and secondary screening, the inventor of the invention obtains 924 strains of mycoplasma pneumoniae.

3. Gene identification of mycoplasma filiformis goat subspecies

Genomic DNAs of bacterial solutions of 924 mycoplasma pneumoniae strains are respectively extracted and used as templates, and PCR amplification is carried out by adopting a mycoplasma filiform goat subspecies specific primer pair (consisting of LC-F: 5'-CAATCCAGATCATAAAAAACCT-3' and LC-R: 5'-CTCCTCATATTCCCCTAGAA-3') to obtain PCR amplification products.

Reaction conditions are as follows: pre-denaturation at 94 ℃ for 3 min; pre-denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 45sec, extension at 72 ℃ for 45sec, 30 cycles; extension at 72 ℃ for 2 min.

If the PCR amplification product of a certain mycoplasma pneumoniae strain contains a 1049bp DNA fragment, the mycoplasma pneumoniae strain is a mycoplasma filiformis goat subspecies.

The 209 mycoplasma pneumoniae strains are identified as mycoplasma filiformis goat subspecies.

4. Gene identification of mycoplasma ovipneumoniae

Genomic DNAs of bacterial solutions of 924 M.pneumoniae strains were extracted, respectively, and PCR amplification was performed using M.pneumoniae specific primer pairs (consisting of Lm-F: 5'-TGAACGGAATATGTTAGCTT-3' and Lm-R: 5'-GACTTCATCCTGCACTCTGT-3') using the extracted genomic DNAs as templates to obtain PCR amplification products.

If the PCR amplification product of a certain Mycoplasma pneumoniae strain contains a 361bp DNA fragment, the Mycoplasma pneumoniae strain is Mycoplasma ovipneumoniae.

The 592 mycoplasma pneumoniae strain was identified as mycoplasma ovipneumoniae.

Example 2 screening and determination of inactivated vaccine Strain and test Strain

First, form observation

A. Morphological observation of Mycoplasma filiformis goat subspecies

1. 5 Mycoplasma filiformis goat subspecies selected in step 3 of example 1 were selected and designated GT-01, GT-02, GT-03, GT-04, and GT-05 in this order, and were obtained from Nemeng Wuyuan county, Chongqing, Guizhou, Xinjiang Uygur autonomous region, and Nemeng Erdos, respectively.

2. The Mycoplasma filiformis goat subspecies (GT-01, GT-02, GT-03, GT-04, or GT-05) were stained with Dienes and observed under a microscope (200X) for colony morphology.

The results of the experiments are shown in FIG. 1(A-E are GT-01, GT-02, GT-03, GT-04 and GT-05 strains in this order). The results show that the colony sizes of 5 mycoplasma filiformis goat subspecies are different, and the diameters are 300-1800 mu m; the colonies are blue, have obvious central umbilicus and do not fade within 30 min. Therefore, the GT-01 strain, GT-02 strain, GT-03 strain, GT-04 strain and GT-05 strain all match the morphological characteristics of the Mycoplasma filiformis goat subspecies.

B. Morphological observation of mycoplasma ovipneumoniae

1. 5 mycoplasma ovipneumoniae selected in the step 4 of example 1 were selected, and named as SH-01 strain, SH-02 strain, SH-03 strain, SH-04 strain and SH-05 strain in sequence, which were respectively derived from Chongqing, Nemenghehote, Xinjiang Uygur autonomous region, Nemengyuan county and NemengErdos.

2. Mycoplasma ovipneumoniae (SH-01, SH-02, SH-03, SH-04, or SH-05) was stained with Dienes and then observed under a microscope (200X) for colony morphology.

The results of the experiments are shown in FIG. 2(A-E are SH-01 strain, SH-02 strain, SH-03 strain, SH-04 strain and SH-05 strain in this order). The results show that the colonies of 5 mycoplasma pneumoniae of sheep are blue, have no central umbilicus and do not fade within 30 min. Thus, the SH-01 strain, the SH-02 strain, the SH-03 strain, the SH-04 strain and the SH-05 strain all accord with the morphological characteristics of the mycoplasma ovipneumoniae.

Second, serological characteristics

A. Serological characterization of Mycoplasma filiformis goat subspecies

A slide agglutination test was performed using standard positive sera from the filamentous mycoplasma goat subspecies PG3 to test the serological characteristics of the filamentous mycoplasma goat subspecies (GT-01, GT-02, GT-03, GT-04, or GT-05 strains).

The detection results are as follows: the 5 mycoplasma filiformis goat subspecies can be inhibited by standard positive serum of the mycoplasma filiformis goat subspecies PG3, and an inhibition zone appears on a filter paper sheet and the diameter of the inhibition zone is more than 2 mm; and no inhibition zone appears around the filter paper sheet soaked with the negative serum. The results show that the standard positive serum of the filiform mycoplasma goat subspecies PG3 can inhibit the growth of 5 filiform mycoplasma goat subspecies, and that GT-01 strain, GT-02 strain, GT-03 strain, GT-04 strain and GT-05 strain all have the serological characteristics of the filiform mycoplasma goat subspecies.

B. Serological characterization of Mycoplasma ovipneumoniae

A glass slide agglutination test is carried out by adopting standard positive serum of the mycoplasma ovipneumoniae model strain Y98 to detect the serological characteristics of the mycoplasma ovipneumoniae (SH-01 strain, SH-02 strain, SH-03 strain, SH-04 strain or SH-05 strain).

The detection results are as follows: the 5 mycoplasma ovipneumoniae can be inhibited by standard positive serum of a mycoplasma ovipneumoniae model strain Y98, and an inhibition zone appears on a filter paper sheet and has the diameter of more than 2 mm; and no inhibition zone appears around the filter paper sheet soaked with the negative serum. The result shows that the standard positive serum of the mycoplasma ovipneumoniae model strain Y98 can inhibit the growth of 5 mycoplasma ovipneumoniae, and SH-01 strain, SH-02 strain, SH-03 strain, SH-04 strain and SH-05 strain all have the serological characteristics of the mycoplasma ovipneumoniae.

Third, biochemical characteristic detection

A. Detecting biochemical characteristics of mycoplasma filiformis goat subspecies

Biochemical characteristics of the filamentous mycoplasma goat subspecies (GT-01 strain, GT-02 strain, GT-03 strain, GT-04 strain, GT-05 strain or the filamentous mycoplasma goat subspecies PG3 strain) were examined.

The results of the biochemical characterization are shown in Table 1(PG3 model strain is strain PG3, a Mycoplasma filiformis subspecies goat). The results showed that the biochemical characteristics of the 5 Mycoplasma filiformis goat subspecies (GT-01, GT-02, GT-03, GT-04, and GT-05) were completely consistent with those of the Mycoplasma filiformis goat subspecies PG 3.

Therefore, GT-01 strain, GT-02 strain, GT-03 strain, GT-04 strain and GT-05 strain all meet the biochemical characteristics of the mycoplasma filiformis goat subspecies.

TABLE 1 Biochemical characteristics of Mycoplasma filiformis goat subspecies

Note: "+" indicates positive, "-" indicates negative, and β indicates β hemolysis.

B. Detecting biochemical characteristics of mycoplasma ovipneumoniae

The biochemical characteristics of Mycoplasma ovipneumoniae (SH-01 strain, SH-02 strain, SH-03 strain, SH-04 strain, SH-05 strain or Mycoplasma ovipneumoniae model strain Y98) were examined.

The results of the biochemical characterization are shown in Table 2 (the model strain Y98 is Mycoplasma ovipneumoniae model strain Y98). The results showed that 5 strains of Mycoplasma ovipneumoniae (SH-01, SH-02, SH-03, SH-04 and SH-05) had biochemical characteristics completely identical to those of M.ovipneumoniae model strain Y98.

Thus, the SH-01 strain, the SH-02 strain, the SH-03 strain, the SH-04 strain and the SH-05 strain all accord with the biochemical characteristics of the mycoplasma ovipneumoniae.

TABLE 2 Biochemical characteristics of Mycoplasma ovipneumoniae

Fourth, viable bacteria titer detection

A. Viable bacteria titer detection of mycoplasma filiformis goat subspecies

Viable cell titers of the Mycoplasma filiformis goat subspecies (GT-01, GT-02, GT-03, GT-04, or GT-05 strains) were determined. The specific method comprises the following steps: inoculating filamentous mycoplasma goat subspecies to a mycoplasma liquid culture medium, and culturing in a constant-temperature incubator at 37 ℃ to obtain a seed solution; performing 10-fold serial dilution on the seed liquid by using a mycoplasma liquid culture medium to obtain a dilution liquid; all dilutions were incubated at 37 ℃ for 10 days, and the highest dilution at which the medium color changed was taken as the viable titer of the mycoplasma.

As a result, the viable cell titers of GT-01, GT-02, GT-03, GT-04 and GT-05 strains were all 1X 10⁹ccu/mL。

B. Viable bacteria titer detection of mycoplasma ovipneumoniae

The titer of viable bacteria of Mycoplasma ovipneumoniae (SH-01 strain, SH-02 strain, SH-03 strain, SH-04 strain or SH-05 strain) is detected. The specific method comprises the following steps: inoculating the mycoplasma ovipneumoniae to a mycoplasma liquid culture medium, and culturing in a constant-temperature incubator at 37 ℃ to obtain a seed solution; performing 10-fold serial dilution on the seed liquid by using a mycoplasma liquid culture medium to obtain a dilution liquid; all dilutions were incubated at 37 ℃ for 10 days, and the highest dilution at which the medium color changed was taken as the viable titer of the mycoplasma.

The results showed that the viable cell titers of SH-01, SH-02, SH-03, SH-04 and SH-05 strains were 1X 10⁹ccu/mL。

Fifth, immunological cross reaction with other strains of the same subspecies

A. Immunological cross reaction between filiform mycoplasma goat subspecies and other bacterial strains of the same subspecies

1. Serum 1 was prepared by immunizing rabbits with filamentous mycoplasma goat subspecies (GT-01, GT-02, GT-03, GT-04, or GT-05).

2. Serum 2 was prepared by immunizing rabbits with each of the other mycoplasma filiformis goat subspecies isolated in example 1.

3. Rabbits were immunized with the Mycoplasma filiformis goat subspecies PG3 strain and bled from the neck to prepare serum 3.

4. Serum 1 and the serum to be tested (serum 2 or serum 3) were subjected to immunological cross-reaction.

The experimental result shows that the cross reaction rate of the serum of the mycoplasma filiform goat subspecies (GT-01 strain, GT-02 strain, GT-03 strain, GT-04 strain or GT-05 strain) and other mycoplasma filiform goat subspecies strains is 85-97 percent; the cross-reactivity with the Mycoplasma filiformis goat subspecies PG3 strain was 100%. Therefore, the GT-01 strain, the GT-02 strain, the GT-03 strain, the GT-04 strain and the GT-05 strain have high immunological cross reactivity, wherein the GT-01 strain has the highest immunological cross protection rate, and provides an advantageous strain for vaccine development. The GT-01 strain was selected for subsequent experiments.

B. Immunological cross reaction of mycoplasma ovipneumoniae with other strains of the same subspecies

1. Serum 1 was prepared by immunizing a rabbit with Mycoplasma ovipneumoniae (SH-01, SH-02, SH-03, SH-04, or SH-05 strain).

2. Serum 2 was prepared by immunizing rabbits with each of the other mycoplasma ovipneumoniae isolates isolated in example 1.

3. The rabbit was immunized with the mycoplasma ovipneumoniae model strain Y98, and serum 3 was prepared by cervical bleeding.

The experimental result shows that the cross reaction rate of the serum of the mycoplasma ovipneumoniae (SH-01 strain, SH-02 strain, SH-03 strain, SH-04 strain or SH-05 strain) and other mycoplasma ovipneumoniae strains is 87-98 percent; the cross-reactivity with M.ovipneumoniae strain Y98 was 100%. Therefore, the SH-01 strain, the SH-02 strain, the SH-03 strain, the SH-04 strain and the SH-05 strain have high immunological cross reactivity, and the SH-01 strain has the highest immunological cross protection rate, so that the strain provides an advantageous strain for vaccine development. SH-01 strain was selected for subsequent experiments.

Sixthly, toxicity test

A. Toxicity test of GT-01 strain on goat

The Minimum Infection Dose (MID) of GT-01 strain to goats was determined by intratracheal injection of GT-01 strain solutions at different concentrations (intratracheal injection of goats with PBS buffer pH7.0 and 0.01mol/L as a control).

The results are shown in Table 3: the minimum infection dose of the GT-01 strain to goats was 5X 10⁹ccu. The GT-01 strain has high toxicity, and provides a dominant strain with strong toxicity for the strain used for detection.

TABLE 3 toxicity test of GT-01 strain on goat

B. Toxicity test of SH-01 strain on goat

The Minimum Infection Dose (MID) of SH-01 strain to goats was detected by intratracheal injection of SH-01 strain solutions of different concentrations (intratracheal injection of goats with PBS buffer (pH7.0) and 0.01mol/L as a control).

The results are shown in Table 4: the minimum infection dose of the SH-01 strain to the goat is 8 multiplied by 10⁹ccu. The SH-01 strain has high toxicity, and provides an advantageous strain with strong toxicity for the strain for inspection.

TABLE 4 toxicity test of SH-01 strain on goat

Seventhly, immunogenicity

A. Immunogenicity of the GT-01 Strain

1. Inoculating GT-01 strain with mycoplasma liquid culture medium, culturing at 37 deg.C to obtain the product with concentration of 2 × 10⁸ccu/mL of GT-01 strain antigen stock solution.

2. And (3) after the step 1 is completed, mixing the GT-01 strain antigen stock solution and the SP01 adjuvant in equal volume, and emulsifying to obtain the inactivated vaccine.

3. After step 2 is completed, the inactivated vaccine is injected subcutaneously into the immunized goat.

4. And (3) collecting blood 21 days after the step 2 is finished, and performing indirect hemagglutination to determine the antibody titer.

5. 21 days after the completion of the step 2, the goat was challenged with the GT-01 strain (the dose of the drug for challenge was 5X 10)⁹ccu), continuously observing for 21 days after attacking, and recording the disease condition of the goat.

The results show that the geometric mean value of the antibody titer produced by the immunized goat is 1:74, the immune goat can obtain complete protection after the toxic attack. The GT-01 strain has good immunogenicity and immune protection, and provides a dominant vaccine strain for the strain for vaccine production.

B. Immunogenicity of SH-01 Strain

1. Inoculating SH-01 strain to mycoplasma liquid culture medium, culturing at 37 deg.C to obtain the product with concentration of 2 × 10⁸ccu/mL of SH-01 strain antigen stock solution.

2. And (3) after the step 1 is finished, mixing the SH-01 strain antigen stock solution and the SP01 adjuvant in equal volume, and emulsifying to obtain the inactivated vaccine.

The results show that the geometric mean value of the antibody titer produced by the immunized goat is 1: 64, the immune goat can obtain complete protection after the toxin attack. The SH-01 strain has good immunogenicity and immune protection, and provides a dominant vaccine strain for a vaccine production strain.

The SH-01 strain has been preserved in China general microbiological culture Collection center (CGMCC, address No. 3 of Xilu No.1 of Beijing, Chaoyang, North Cheng) in 19.09.2018, and the preservation number is CGMCC No. 16503. The SH-01 strain is called Mycoplasma ovipneumoniae (Mycoplasma ovipneumoniae) SH-01CGMCC No. 16503.

The GT-01 strain has been deposited in China general microbiological culture Collection center (CGMCC, address No. 3 Xilu No.1 Beijing, Chaoyang, China) in 19.09.2018, and the deposition number is CGMCC No. 16504. The GT-01 strain is called as filamentous Mycoplasma goat subspecies (Mycoplasma mycoidssubsp. capri) GT-01 CGMCCNo.16504.

Example 3 expression of MOMP protein (Chlamydophila psittaci major outer Membrane protein)

Construction of recombinant plasmid pET-22b-MOMP

1. Since the MOMP gene of Chlamydophila psittaci is highly conserved among species, the MOMP gene (GenBank: L25436.1) represented typically by Cps was selected for codon optimization. Artificially synthesizing a nucleotide sequence (namely MOMP gene) shown as a sequence 1 in the sequence table after codon optimization.

2. Taking the nucleotide sequence of the MOMP gene synthesized in the step 1 as a template and taking 5' -TATTCATATGTTGCCTGTAGGGAACCCAGCT-3 '(recognition sites for the restriction enzymes NdeI are underlined) and 5' -GCGCCTCGAGCTAGAATCTGAATTGAGCATT-3' (recognition sites for restriction enzyme XhoI are underlined) were subjected to PCR amplification to obtain a PCR amplification product of about 1130 bp.

3. Taking the PCR amplification product obtained in the step 2, carrying out enzyme digestion by using restriction enzymes NdeI and XhoI, and recovering an enzyme digestion product of about 1130 bp.

4. The vector pET-22b (+) (product of Novagen Co.) was digested with restriction enzymes NdeI and XhoI, and a 5363bp vector backbone was recovered.

5. And connecting the enzyme digestion product with a vector skeleton to obtain the recombinant plasmid pET-22 b-MOMP.

The recombinant plasmid pET-22b-MOMP was sequenced. According to the sequencing result, the structure of the recombinant plasmid pET-22b-MOMP is described as follows: the small DNA fragment between the recognition sequence of restriction enzyme NdeI and the recognition sequence of restriction enzyme XhoI in the vector pET-22b (+) is replaced by a DNA molecule with the nucleotide sequence shown in the sequence 1 of the sequence table. The recombinant plasmid pET-22b-MOMP expresses MOMP protein shown in a sequence 2 in a sequence table.

Expression of the two, MOMP proteins

1. And (3) transforming the recombinant plasmid pET-22b-MOMP into escherichia coli DH5 alpha competent cells to obtain the recombinant escherichia coli.

2. A single clone of the recombinant E.coli was inoculated into 5mL of LB liquid medium (containing 50. mu.g/mL of ampicillin), and cultured overnight at 37 ℃ with shaking at 250rpm to obtain a culture broth.

3. After completion of step 2, the culture broth was inoculated (inoculation ratio 1:100) to 500mL of LB liquid medium (containing 50. mu.g/mL of ampicillin) and cultured at 37 ℃ and 250rpm with shaking to OD_600nmThe concentration of IPTG was adjusted to 1mM in the system at a value of 0.4-0.6, followed by shaking culture at 250rpm at 16 ℃ for 16 hours and centrifugation at 5000g for 10min at 4 ℃ to collect the cells.

4. The cells obtained in step 3 were washed with pre-cooled purified water 2 times, and then resuspended in 30mL of a 50mM Tris-Cl buffer solution containing 1mM EDTA, 1mM PMSF, 10% (v/v) glycerol and pH 7.4.

5. After step 4, the resuspension solution was subjected to ultrasonication using a cell disruptor (5 s intermittent at 10KG, 5s disruption, 5min total disruption), then centrifuged at 13000rpm for 15min at 4 ℃ and the precipitate and supernatant were collected.

6. The pellet and supernatant collected in step 5 were subjected to SDS-PAGE.

The SDS-PAGE results are shown in FIG. 3(1 is supernatant, 2 and 3 are precipitates, and 4 is protein Marker). The results showed that the expression amount of MOMP protein was 23%, and MOMP protein was mainly present in the form of inclusion bodies.

Example 4 preparation and assay of SH-01 inactivated Strain antigen stock solution

Preparation of SH-01 seed liquid

And inoculating the SH-01 strain to a mycoplasma liquid culture medium, and culturing in a constant-temperature incubator at 37 ℃ to obtain SH-01 seed liquid. In the SH-01 seed liquid, the concentration of SH-01 is 1 multiplied by 10⁹ccu/mL。

II, preparation of SH-01 inactivated thallus antigen stock solution

1. Taking 10L fermentation tank, adding basic culture solution with volume of 4.5L, and autoclaving at 121 deg.C for 15 min.

2. After the step 1 is completed, when the temperature is reduced to 37-38 ℃, adding 1.2L of horse serum, 300mL of auxiliary liquid and 120 ten thousand units of Penicilin, and uniformly mixing. Then 600mLSH-01 seed liquid is added, and fermentation culture is carried out for 72 h.

The fermentation conditions were: 37 ℃; the tank pressure is kept at 0.05 MPa; the pH value of the aqueous solution of 10% (v/v) ammonia water is automatically adjusted to be kept at 7.8; the initial rotation speed is 200rpm, the dissolved oxygen continuously decreases along with the growth of thalli, and the dissolved oxygen is adjusted by automatically adjusting the rotation speed and the ventilation capacity to keep the dissolved oxygen at 20-30%.

3. After the step 2 is finished, adding formaldehyde solution (for inactivation) according to 0.2% (v/v) of the volume of the fermentation liquid, and culturing at 25 ℃ for 10h to obtain the inactivated bacterial liquid.

4. And (3) taking the inactivated bacterial liquid obtained in the step (3), firstly carrying out ultrafiltration concentration (100KD) purification, then concentrating by 20 times, and collecting to obtain SH-01 inactivated bacterial antigen stock solution.

The SH-01 inactivated thallus antigen stock solution can be stored for less than 6 months at the temperature of 2-8 ℃.

Third, detection of SH-01 inactivated thallus antigen stock solution

1. Sterility test of SH-01 inactivated thallus antigen stock solution

And (3) taking a TG tubule, inoculating 0.2mL of SH-01 inactivated thalli antigen stock solution, culturing for 7 days at 37 ℃ or 25 ℃, and observing whether bacteria grow.

The results showed that the TG tubules were aseptically grown. Therefore, SH-01 inactivated thallus antigen stock solution is sterile.

2. Determination of viable bacteria titer of SH-01 inactivated thallus antigen stock solution

And (3) taking the SH-01 seed solution prepared in the step one, and performing 10-fold serial dilution on the seed solution by using a mycoplasma liquid culture medium to obtain a diluted solution. All dilutions were incubated at 37 ℃ for 10 days, and the highest dilution that could undergo a color change (from red to yellow) was the viable titer.

The result shows that the live bacteria titer of the SH-01 inactivated thallus antigen stock solution is not less than 1 multiplied by 10⁸ccu/mL。

Example 5 preparation and assay of GT-01 inactivated thallus antigen stock solution

1. The SH-01 strain in the first and second steps of example 4 was replaced with GT-01 strain, and the other steps were not changed to obtain a GT-01 inactivated somatic antigen stock solution.

2. Assay for GT-01 inactivated somatic antigen stock solution

(1) Sterility testing of GT-01 inactivated thallus antigen stock solution

And (3) taking a TG tubule, inoculating 0.2mL of GT-01 inactivated thalli antigen stock solution, culturing for 7 days at 37 ℃ or 25 ℃, and observing whether bacteria grow.

The results showed that the TG tubules were aseptically grown. Therefore, the GT-01 inactivated thallus antigen stock solution is sterile.

(2) Live bacteria titer determination of GT-01 inactivated thallus antigen stock solution

And (3) taking the GT-01 seed solution prepared in the step (1), and performing 10-fold serial dilution on the GT-01 seed solution by using a mycoplasma liquid culture medium to obtain a dilution solution. All dilutions were incubated at 37 ℃ for 10 days, and the highest dilution that could undergo a color change (from red to yellow) was the viable titer.

The results showed that the live titer of the GT-01 inactivated bacterial antigen stock solution was 1X 10⁹ccu/mL。

Example 6 preparation and assay of MOMP protein antigen stock

Preparation of recombinant escherichia coli seed liquid

1. A single clone of the recombinant E.coli strain obtained in step two of example 3 was inoculated into 5mL of LB liquid medium (containing 50. mu.g/mL of ampicillin), and cultured overnight at 37 ℃ and 250rpm with shaking to obtain a culture broth.

2. After completing step 1, the culture broth was inoculated (inoculation ratio 1:100) to 500mL of LB liquid medium (containing 50. mu.g/mL ampicillin) and cultured with shaking at 37 ℃ and 250rpm to obtain a recombinant large intestineBacillus seed liquid. OD of recombinant Escherichia coli seed liquid_600nmThe value is 0.4-0.6.

Preparation of MOMP protein antigen stock solution

1. Taking 10L fermentation tank, adding LB liquid culture medium with volume of 5L, and autoclaving at 121 deg.C for 15 min.

2. After the step 1 is finished, adding ampicillin according to the final concentration of 100 mug/mL when the temperature is reduced to 37-38 ℃, and uniformly mixing; then 250mL of recombinant Escherichia coli seed solution (the inoculum size is 5%) is added, and fermentation culture is carried out for 4h at 37 ℃.

The fermentation conditions were: the tank pressure is kept at 0.05 MPa; the pH value of the aqueous solution of 10% (v/v) ammonia water is automatically adjusted to be kept at 7.2; the initial rotating speed is 200rpm, the dissolved oxygen quantity is continuously reduced along with the growth of thalli, and the dissolved oxygen quantity is adjusted by automatically adjusting the rotating speed and the ventilation quantity, so that the dissolved oxygen quantity is kept at 25 percent.

3. After the step 2 is finished, adding IPTG (isopropyl-beta-thiogalactoside) to enable the concentration of the IPTG in a fermentation system to be 1mM, and carrying out fermentation culture at 37 ℃ for 6h to obtain a zymophyte liquid.

4. And (4) centrifuging the zymocyte liquid obtained in the step (3) at 4 ℃ at 8000r/min for 15min, and collecting thalli.

5. Weighing the thallus collected in the step 4, adding 20ml of PBS (phosphate buffer solution) with the pH value of 7.0 and the concentration of 0.01mM into 1g of thallus, and carrying out heavy suspension to obtain the heavy suspension.

6. And (3) taking the heavy suspension obtained in the step (5), crushing the heavy suspension for 4 times by using a high-pressure homogenizer at the pressure of 700bar, centrifuging for 20min at the temperature of 4 ℃ at 10000r/min, and collecting the precipitate. The collected precipitate was washed twice.

7. Taking the precipitate washed in the step 6, and adding an inclusion body dissolving solution (Tris-HCl buffer solution containing 8M urea and 5mM DTT, pH8.0 and 0.02M) according to the proportion of 50-100 mL/g for fully dissolving; then, the mixture was centrifuged at 10000rpm for 20min, and the supernatant was collected. The supernatant was the denatured MOMP protein.

8. Taking the supernatant collected in the step 7, performing gradient dialysis by PBS buffer solutions with different urea contents (8mol/L, 6mol/L, 4mol/L, 2mol/L and 0mol/L) and pH7.0 and 0.01mol/L respectively to perform protein renaturation, collecting renatured protein solution, and filtering by using a filter with the aperture of 0.22 mu m to obtain the protein solution.

The protein liquid is stored at-20 deg.C for use, and the shelf life is no more than 6 months.

9. And (3) adding a formaldehyde solution (with the aim of inactivation) with the final concentration of 0.1% of formaldehyde into the protein solution obtained in the step (8), and culturing at 25 ℃ for 25h to obtain MOMP protein antigen stock solution.

Detection of MOMP protein antigen stock solution

1. Sterility testing of MOMP protein antigen stock

Inoculating 0.2 mM LMOMP protein antigen stock solution to TG tubule, culturing at 37 deg.C or 25 deg.C for 7 days, and observing whether bacteria grow.

The results showed that the TG tubules were aseptically grown. Therefore, the MOMP protein antigen stock solution is sterile.

2. Detection of protein content of MOMP protein antigen stock solution

Protein content was determined using BCA assay.

The result shows that the protein content in the MOMP protein antigen stock solution is 3.0 mg/mL.

Four, immunogenicity detection of MOMP proteins

(1) And (3) taking the protein solution obtained in the step (8) in the step (II), and diluting the protein solution with PBS (phosphate buffer solution) with the pH value of 7.0 and the concentration of 0.01mol/L to obtain MOMP protein diluent.

(2) And mixing 1 volume part of MOMP protein diluent and 1 volume part of ISA201 adjuvant to obtain the MOMP protein vaccine. In the MOMP protein vaccine, the content of MOMP protein is 0.3 mg/mL.

(3) 10 female goats aged 2-4 years were randomly divided into two groups, 5 in each group, of the test group and the control group. Then the following treatment is carried out:

test groups: on test day 1, each goat was injected subcutaneously with 3ml lmomp protein vaccine; on the 21 st day of the test, blood was collected intravenously and then specific antibodies were detected by indirect ELISA; on the 45 th day of the experiment, 2mL of Chlamydia psittaci CG1 bacterial solution (3X 10) was intraperitoneally injected⁹ELD₅₀) And (5) carrying out toxicity attack and observing until the lambing is finished.

Control group: on the 1 st day of the experiment, each goat was injected subcutaneously with 3ml of PBS buffer at pH7.0 and 0.01 mol/L; on the 21 st day of the test, blood was collected intravenously and then specific antibodies were detected by indirect ELISA; on the 45 th day of the experiment, intraperitoneal injection2mL of Chlamydia psittaci CG1 bacterial liquid (3X 10)⁹ELD₅₀) And (5) carrying out toxicity attack and observing until the lambing is finished.

The result shows that the antibody titer of each goat in the test group is more than 1:4222, and the goat can be protected by 5/5 after toxin attack; the antibody titer of each goat in the control group is below 1:200, and the goat can be protected by 0/5 after being attacked by toxin. Thus, the MOMP protein has good immunogenicity.

Example 7 preparation of a bivalent inactivated vaccine against Mycoplasma capricolumni pneumonia and Chlamydia psittaci disease

1. Taking SH-01 inactivated thallus antigen stock solution, and diluting the stock solution with PBS buffer solution with the pH value of 7.0 and the concentration of 0.01mol/L to obtain SH-01 diluent solution; the concentration of SH-01 strain in SH-01 dilution was 1.2X 10⁹ccu/mL. Taking GT-01 inactivated thallus antigen stock solution, diluting with PBS buffer solution with pH7.0 and 0.01mol/L to obtain GT-01 diluent solution; the concentration of the GT-01 strain in the GT-01 dilution was 1.2X 10⁹ccu/mL. Taking MOMP protein antigen stock solution, and diluting with PBS buffer solution with pH7.0 and 0.01mol/L to obtain MOMP protein diluent; the concentration of MOMP protein in the MOMP protein dilution was 1.8 mg/mL.

2. And (3) after the step 1 is completed, mixing 1 volume part of SH-01 diluent, 1 volume part of GT-01 diluent and 1 volume part of MOMP protein diluent to obtain a goat mycoplasma pneumonia and psittacosis Chlamydia bigeminy inactivated vaccine whole antigen mixed solution. Placing the combined inactivated vaccine whole antigen mixed solution of the mycoplasma pneumoniae of the goats and the chlamydia psittaci disease at 2-8 ℃ and storing for later use.

3. Mixing 1 volume part of the goat mycoplasma pneumonia and psittacosis combined inactivated vaccine complete antigen mixed solution and 1 volume part of adjuvant (ISA201, SP01, MF59 or aluminum hydroxide), and emulsifying to obtain the goat mycoplasma pneumonia and psittacosis combined inactivated vaccine. The vaccine is packaged in a proper amount and stored at 2-8 ℃.

Example 8, example 7 preparation of goat mycoplasma pneumonia and Chlamydia psittaci bivalent inactivated vaccine safety test

First, hemolytic test

1. A guinea pig with a weight of about 350g was selected, and 1mL of fresh guinea pig blood was collected, washed 3 times with PBS buffer solution of pH7.0 and 0.01mol/L, and the volume of blood cells was recovered with PBS buffer solution of pH7.0 and 0.01mol/L and diluted 10-fold to obtain guinea pig blood cells.

2. The goat mycoplasma pneumonia and psittacosis combined inactivated vaccine prepared in example 7 was diluted 2-fold, 4-fold, or 8-fold with PBS buffer solution (pH7.0, 0.01 mol/L) to obtain a vaccine dilution.

3. And (3) after the step 2 is finished, adding the guinea pig blood cells prepared in the step 1 into the vaccine diluent, and standing at room temperature for 8 hours. Whether the blood cells were lysed or ruptured was visually observed.

4. After completion of step 3, the system which completed step 3 was taken, centrifuged at 400rpm for 5min, and the supernatant was collected and absorbance was measured at 570 nm.

The results show that no rupture of the blood cells occurred and no hemolysis occurred. Therefore, the inactivated vaccine for mycoplasma capricolum pneumonia and chlamydia psittaci prepared in example 7 can not lyse erythrocytes, and has no hemolytic reaction.

Second, toxicity test

1. Toxicity test in rabbits

10 rabbits with the weight of 2.0-3.0 kg are taken and randomly divided into a test group and a control group (5 rabbits in each group), and then the following treatment is carried out:

test groups: 3mL of the goat mycoplasma pneumonia and Chlamydia psittaci combined inactivated vaccine prepared in example 7 was intramuscularly injected into each rabbit, and then the activity status, weight change and survival rate of the rabbits were observed for 10 consecutive days. After 10 days, the patient was sacrificed for anatomical examination.

Control group: each rabbit was injected intramuscularly with 3mL of PBS buffer (pH7.0, 0.01 mol/L), and then the activity status, weight change and survival rate of the rabbits were observed for 10 consecutive days. After 10 days, the patient was sacrificed for anatomical examination.

The results showed that the rabbits of the test group and the control group all survived without adverse symptoms such as piloerection, listlessness, decreased appetite, and slow movement, and the body weight was increased. After 10 days, the rabbits were sacrificed and examined for dissection, and the pathological changes of the organs were not observed in the rabbits of the test group and the control group. Therefore, the combined inactivated vaccine for mycoplasma capricolum pneumonia and chlamydia psittaci disease prepared in example 7 is safe for rabbits.

2. Beagle dog toxicity test

10 Beagle dogs weighing 8-10 kg were randomly divided into test and control groups (5 dogs each) and then treated as follows:

test groups: each Beagle dog was intramuscularly injected with 15mL of the combined inactivated vaccine of mycoplasma capricolum pneumonia and chlamydia psittaci prepared in example 7, and then behavior, body weight and survival rate of the Beagle dog were observed for 14 consecutive days. After 14 days, the patient was sacrificed for anatomical examination.

Control group: the Beagle dogs were each intramuscularly injected with 15mL of 0.01mol/L PBS buffer at pH7.0, and then observed for behavior, body weight and survival rate changes for 14 consecutive days. After 14 days, the patient was sacrificed for anatomical examination.

The result shows that the Beagle dogs in the test group have no toxic reaction, normal behaviors and no death, and have no difference with the Beagle dogs in the control group, and the weight average of all dogs is increased; after 14 days, Beagle dogs were sacrificed and examined for dissection, and no pathological changes in the organs were observed in the Beagle dogs in the test and control groups. Therefore, the inactivated bivalent vaccine for mycoplasma capricolum pneumonia and chlamydia psittaci disease prepared in example 7 has no acute toxicity reaction and is safe for Beagle dogs.

3. Goat toxicity test

10 healthy susceptible ewes, 3 months old gestational age, were randomly divided into test and control groups (5 per group) and then treated as follows:

test groups: 6mL of the goat mycoplasma pneumoniae and Chlamydia psittaci combined inactivated vaccine prepared in example 7 was injected bilaterally and subcutaneously into the neck of each goat, and then the injection site and the systemic state of the goat were observed for 30 consecutive days.

Control group: each goat was subcutaneously injected into 6ml of PBS buffer (pH 7.0, 0.01 mol/L) in the neck at both sides, and then the injection site and the systemic state of the goat were observed for 30 consecutive days.

The results show that the goat mental states of the test group and the control group are good, and no obvious local or systemic adverse reaction exists. Therefore, the inactivated vaccine for mycoplasma capricolum pneumonia and Chlamydia psittaci disease prepared in example 7 has no acute toxicity reaction and is safe for goats.

Third, allergy test study

21 Hartley guinea pigs weighing 250-350 g were randomly divided into a first group (5), a second group (5), a third group (5), a fourth group (3) and a fifth group (3), and then treated as follows:

a first group: each Hartley guinea pig was subcutaneously inoculated with 0.5mL of the goat mycoplasma pneumoniae and psittacosis combined inactivated vaccine prepared in example 7, and the status of the animals was observed 30 minutes and 3 days after the injection.

Second group: each Hartley guinea pig was intraperitoneally injected with 0.5mL of the goat mycoplasma pneumoniae and Chlamydia psittaci bivalent inactivated vaccine prepared in example 7, and the state of the animals was observed 30 minutes and 3 days after the injection.

Third group: each Hartley guinea pig was intraperitoneally injected with 1mL of the goat mycoplasma pneumoniae and Chlamydia psittaci bivalent inactivated vaccine prepared in example 7, and the state of the animals was observed 30 minutes and 3 days after the injection.

And a fourth group: each Hartley guinea pig was intraperitoneally injected with 1mL of physiological saline, and the state of the animals was observed 30 minutes and 3 days after the injection.

And a fifth group: each Hartley guinea pig was intraperitoneally injected with 1mL of human serum albumin, and the status of the animals was observed 30 minutes and 3 days after injection.

The results showed that none of the guinea pigs in the first, second, third and fourth groups died and had no allergic symptoms such as nasal itching, sneezing, dysphoria, dyspnea, shock, spasm and the like. The guinea pigs in the fifth group developed allergic symptoms (the fifth group was a positive control, consistent with expected experimental results). Therefore, the inactivated vaccine for mycoplasma capricolum pneumonia and chlamydia psittaci disease prepared in example 7 has no allergic reaction in animals.

EXAMPLE 9, EXAMPLE 7 evaluation of the Immunity efficacy of the inactivated vaccine for Mycoplasma capricolumni pneumonia and Chlamydia psittaci disease in animals

Immune effect of the one-and two-way inactivated vaccine on healthy susceptible ewes

10 healthy susceptible ewes about 15 days before mating were selected, randomly divided into test groups and control groups (5 in each group), and then treated as follows:

test groups: on the 1 st day of the experiment, 3mL of the goat mycoplasma pneumonia and psittacosis combined inactivated vaccine prepared in example 7 is injected subcutaneously into the neck of each goat; on day 21 of the test, each goat was bled intravenously, sera were separated, the antibody titer of the mycoplasma filiformis goat subspecies and mycoplasma ovipneumoniae was determined by indirect hemagglutination assay, and the antibody titer of chlamydia psittaci was determined by indirect ELISA.

Control group: on the 1 st day of the experiment, 3mL PBS buffer solution with pH7.0 and 0.01mol/L is injected subcutaneously in the neck of each goat; on day 21 of the test, each goat was bled intravenously, sera were separated, the antibody titer of the mycoplasma filiformis goat subspecies and mycoplasma ovipneumoniae was determined by indirect hemagglutination assay, and the antibody titer of chlamydia psittaci was determined by indirect ELISA.

The experimental results are as follows: in the test group, the geometric mean values of the antibody titers of the immune goats against the mycoplasma filiformis goat subspecies and the mycoplasma ovipneumoniae in the test group are respectively 1: 84 and 1:74, the geometric mean value of the antibody titer of the Chlamydia psittaci can reach 1: 4850; in the control group, the antibody titer of the immune goat against mycoplasma ovipneumoniae is not higher than 1:4, and the antibody titer of chlamydia psittaci is not higher than 1: 200. Therefore, the goat mycoplasma pneumonia and psittacosis combined inactivated vaccine prepared in example 7 has a good immune effect on healthy susceptible ewes.

Immune protection effect of bivalent inactivated vaccine after goat immunization

Selecting 30 healthy susceptible ewes about 15 days before hybridization, randomly dividing the ewes into an immune group I, an immune group II, an immune group III, a control group I, a control group II and a control group III (5 in each group), and then treating the ewes as follows:

immunization group I: on the 1 st day of the experiment, 3mL of the goat mycoplasma pneumonia and psittacosis combined inactivated vaccine prepared in example 7 is injected subcutaneously into the neck of each goat; on the 45 th day of the experiment, 10mLGT-01 strain of bacterial liquid (5X 10) was injected into each goat trachea⁹ccu) counteracting toxic materials; continuously observing until the lambing is finished;

immunization group II: on the 1 st day of the experiment, 3mL of the goat mycoplasma pneumonia and psittacosis combined inactivated vaccine prepared in example 7 is injected subcutaneously into the neck of each goat; on the 45 th day of the experiment, 10mLSH-01 strain of bacterial liquid (8X 10) was injected into the trachea of each goat⁹ccu) counteracting toxic materials; continuously observing until the lambing is finished;

immunization group III: on the 1 st day of the experiment, 3mL of the goat mycoplasma pneumonia and psittacosis combined inactivated vaccine prepared in example 7 is injected subcutaneously into the neck of each goat; on day 45 of the experiment, each goat was intratracheally injected with 2mL of Chlamydia psittaci CG1 (3X 10)⁹ELD₅₀) Counteracting toxic substances; continuously observing until the lambing is finished;

control group I: on the 1 st day of the experiment, 3mL PBS buffer solution with pH7.0 and 0.01mol/L is injected subcutaneously in the neck of each goat; on the 45 th day of the experiment, 10mL of GT-01 strain bacterial liquid (5X 10) was injected into each goat trachea⁹ccu) counteracting toxic materials; continuously observing until the lambing is finished;

control group II: on the 1 st day of the experiment, 3mL PBS buffer solution with pH7.0 and 0.01mol/L is injected subcutaneously in the neck of each goat; on the 45 th day of the experiment, 10mLSH-01 strain of bacterial liquid (8X 10) was injected into the trachea of each goat⁹ccu) counteracting toxic materials; continuously observing until the lambing is finished;

control group III: on the 1 st day of the experiment, 3mL PBS buffer solution with pH7.0 and 0.01mol/L is injected subcutaneously in the neck of each goat; on day 45 of the experiment, each goat was intratracheally injected with 2mL of Chlamydia psittaci CG1 (3X 10)⁹ELD₅₀) Counteracting toxic substances; the end of lambing was observed continuously.

In each group, the goat needs to be fed separately after the toxin is attacked, and the goat response after the toxin is closely observed.

The results show that in the immunization group I, the immunization group II and the immunization group III, the goat mycoplasma pneumonia and psittacosis bigeminy inactivated vaccine prepared in the example 7 can protect the GT-01 strain, the SH-01 strain and the psittacosis Chlamydia CG1 from 5/5; in the control group I, the control group II and the control group III, the goat attacked all developed diseases. Therefore, the goat mycoplasma pneumonia and psittacosis combined inactivated vaccine prepared in example 7 produces good immune protection effect after being used for immunizing goats.

Example 10, example 7 preparation of goat mycoplasma pneumonia and Chlamydia psittaci disease bivalent inactivated vaccine in goat in vivo long-acting immune protection evaluation

1. 70 healthy susceptible ewes about 15 days before mating were selected, randomly divided into test groups and control groups (35 in each group), and then treated as follows:

test groups: on the 1 st day of the experiment, 3mL of the goat mycoplasma pneumonia and psittacosis combined inactivated vaccine prepared in example 7 is injected subcutaneously into the neck of each goat; blood is collected intravenously and serum is separated at the 14 th test day, the 21 st test day, the 45 th test day, the 6 th test month, the 8 th test month and the 10 th test month respectively, the antibody titer of the mycoplasma filiformis goat subspecies and the mycoplasma ovipneumoniae is detected by adopting an indirect hemagglutination test, and the antibody titer of the chlamydia psittaci is detected by adopting an indirect ELISA.

Control group: on the 1 st day of the experiment, 3mL PBS buffer solution with pH7.0 and 0.01mol/L is injected subcutaneously in the neck of each goat; blood is collected intravenously and serum is separated at the 14 th test day, the 21 st test day, the 45 th test day, the 6 th test month, the 8 th test month and the 10 th test month respectively, the antibody titer of the mycoplasma filiformis goat subspecies and the mycoplasma ovipneumoniae is detected by adopting an indirect hemagglutination test, and the antibody titer of the chlamydia psittaci is detected by adopting an indirect ELISA.

2. Selecting 30 healthy susceptible ewes about 15 days before mating, randomly dividing the ewes into a test 1 group, a test 2 group, a test 3 group, a control 1 group, a control 2 group and a control 3 group (5 in each group), and then treating the ewes as follows:

test 1 group: on the 1 st day of the experiment, 3mL of the goat mycoplasma pneumonia and psittacosis combined inactivated vaccine prepared in example 7 is injected subcutaneously into the neck of each goat; on the 45 th day of the experiment, 10mL of GT-01 strain bacterial liquid (5X 10) was injected into each goat trachea⁹ccu) counteracting toxic materials; feeding in isolation after attacking, observing and recording goat protection and disease occurrence; continuously observing for 30 days;

test 2 groups: on the 1 st day of the experiment, 3mL of the goat mycoplasma pneumonia and psittacosis combined inactivated vaccine prepared in example 7 is injected subcutaneously into the neck of each goat; intratracheal injection of each goat on day 45 of the experimentBacterial liquid of 10mLSH-01 strain (5X 10)⁹ccu) counteracting toxic materials; feeding in isolation after attacking, observing and recording goat protection and disease occurrence; continuously observing for 30 days;

run 3 groups: on the 1 st day of the experiment, 3mL of the goat mycoplasma pneumonia and psittacosis combined inactivated vaccine prepared in example 7 is injected subcutaneously into the neck of each goat; on day 45 of the experiment, each goat was intratracheally injected with 2mL of Chlamydia psittaci CG1 (3X 10)⁹ELD₅₀) Counteracting toxic substances; feeding in isolation after attacking, observing and recording goat protection and disease occurrence; continuously observing for 30 days;

control 1 group: on the 1 st day of the experiment, 3mL PBS buffer solution with pH7.0 and 0.01mol/L is injected subcutaneously in the neck of each goat; on the 45 th day of the experiment, 10mL of GT-01 strain bacterial liquid (5X 10) was injected into each goat trachea⁹ccu) counteracting toxic materials; feeding in isolation after attacking, observing and recording goat protection and disease occurrence; continuously observing for 30 days;

control 2 group: on the 1 st day of the experiment, 3mL PBS buffer solution with pH7.0 and 0.01mol/L is injected subcutaneously in the neck of each goat; 10mLSH-01 strain of bacterial liquid (5X 10) is injected into trachea of each goat⁹ccu) counteracting toxic materials; feeding in isolation after attacking, observing and recording goat protection and disease occurrence; continuously observing for 30 days;

control 3 group: on the 1 st day of the experiment, 3mL PBS buffer solution with pH7.0 and 0.01mol/L is injected subcutaneously in the neck of each goat; on day 45 of the experiment, each goat was intratracheally injected with 2mL of Chlamydia psittaci CG1 (3X 10)⁹ELD₅₀) Counteracting toxic substances; feeding in isolation after attacking, observing and recording goat protection and disease occurrence; the observation was continued for 30 days.

3. Selecting 30 healthy susceptible ewes about 15 days before mating, randomly dividing the ewes into a test 1 group, a test 2 group, a test 3 group, a control 1 group, a control 2 group and a control 3 group (5 in each group), and then treating the ewes as follows:

test 1 group: on the 1 st day of the experiment, 3mL of the goat mycoplasma pneumonia and psittacosis combined inactivated vaccine prepared in example 7 is injected subcutaneously into the neck of each goat; in 8 th month or 10 th month of experiment, 10mLGT-01 strain bacterial liquid (5 × 10 strain) is injected into trachea of each goat⁹ccu) counteracting toxic materials; feeding separately after toxin attack, observing and recordingGoat protection and disease incidence; continuously observing for 30 days;

test 2 groups: on the 1 st day of the experiment, 3mL of the goat mycoplasma pneumonia and psittacosis combined inactivated vaccine prepared in example 7 is injected subcutaneously into the neck of each goat; in the 8 th month or 10 th month of experiment, 10mLSH-01 strain of bacterial liquid (5X 10) is injected into trachea of each goat⁹ccu) counteracting toxic materials; feeding in isolation after attacking, observing and recording goat protection and disease occurrence; continuously observing for 30 days;

run 3 groups: on the 1 st day of the experiment, 3mL of the goat mycoplasma pneumonia and psittacosis combined inactivated vaccine prepared in example 7 is injected subcutaneously into the neck of each goat; at month 8 or 10 of the experiment, each goat was intratracheally injected with 2mL of Chlamydia psittaci CG1 (3X 10)⁹ELD₅₀) Counteracting toxic substances; feeding in isolation after attacking, observing and recording goat protection and disease occurrence; continuously observing for 30 days;

control 1 group: on the 1 st day of the experiment, 3mL PBS buffer solution with pH7.0 and 0.01mol/L is injected subcutaneously in the neck of each goat; in the 8 th month or the 10 th month of the experiment, 10mL of GT-01 strain bacterial liquid (5X 10) is injected into each goat trachea⁹ccu) counteracting toxic materials; feeding in isolation after attacking, observing and recording goat protection and disease occurrence; continuously observing for 30 days;

control 2 group: on the 1 st day of the experiment, 3mL PBS buffer solution with pH7.0 and 0.01mol/L is injected subcutaneously in the neck of each goat; in the 8 th month or 10 th month of experiment, 10mLSH-01 strain of bacterial liquid (5X 10) is injected into trachea of each goat⁹ccu) counteracting toxic materials; feeding in isolation after attacking, observing and recording goat protection and disease occurrence; continuously observing for 30 days;

control 3 group: on the 1 st day of the experiment, 3mL PBS buffer solution with pH7.0 and 0.01mol/L is injected subcutaneously in the neck of each goat; at month 8 or 10 of the experiment, each goat was intratracheally injected with 2mL of Chlamydia psittaci CG1 (3X 10)⁹ELD₅₀) Counteracting toxic substances; feeding in isolation after attacking, observing and recording goat protection and disease occurrence; the observation was continued for 30 days.

The experimental results are as follows: the goat after the immunization of the goat inactivated vaccine for mycoplasma capricolum pneumonia and chlamydia psittaci prepared in example 7 starts to produce antibody titer against mycoplasma filiformis goat subspecies, mycoplasma ovipneumoniae and chlamydia psittaci on day 14; day 21 antibody titers reached a peak; effective immune protection is generated on mycoplasma filiformis goat subspecies and mycoplasma ovipneumoniae when the virus is attacked on the 45 th day; the antibody titer is reduced at month 8, but the antibody titer can still effectively protect mycoplasma filamentous goat subspecies and mycoplasma ovipneumoniae; the antibody titer is obviously reduced at the 10 th month and the effective protection can not be generated on the mycoplasma filiform goat subspecies and the mycoplasma ovipneumoniae. The Chlamydia psittaci is attacked at 45 days after immunization, after attacking, the born lambs are observed, and the protection and disease incidence of the goats are observed and recorded. Therefore, effective protection is observed for nearly 4 months in the priming process, but by 8 months and 10 months of immunization, the chlamydia psittaci challenge is lost because the pregnant sheep already lambed. But in terms of antibody titer, the titer still reaches the titer level of effective protection. It can be seen that the efficacy of the combined inactivated vaccine for mycoplasma capricolum pneumonia and chlamydia psittaci disease prepared in example 7 can last up to 8 months.

<110> inner Mongolia Huaxi Biotech Ltd

<120> bivalent inactivated vaccine for mycoplasma pneumonia and chlamydia psittaci of goats and preparation method thereof

<160>2

<170>PatentIn version 3.5

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<211>1110

<212>DNA

<213> Artificial sequence

<220>

<223>

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ttgcctgtag ggaacccagc tgaaccaagt ttattaatcg atggcactat gtgggaaggt 60

gcttcaggtg atccttgcga tccttgctct acttggtgtg atgctatcag catccgcgca 120

ggatactacg gagattatgt tttcgatcgt gtattaaaag ttgatgtgaa taaaactttc 180

agcggcattg gcaagaaacc cacaggatcc tctccaaatg actttaaaaa tgctgaagat 240

agacccaacg tcgcttatgg cagacatttg caagactccg aatggtttac aaatgcagct 300

ttcttagcgt taaatatctg ggatcgtttt gatattttct gcacattagg cgcttctaat 360

gggtacttca aagctagttc tgcggcattc aatctcgttg gtttgattgg tgttaaagga 420

agctccttaa caaatgacca acttcccaac gtagccatca ctcaaggcgt tgttgagttt 480

tacacagata caacgttctc ttggagcgta ggtgcacgtg gagctctatg ggaatgtggt 540

tgcgcaactt taggagctga attccaatac gctcaatcta atcctaaaat tgaaatgttg 600

aatgtaatct ccagcccagc acaatttgtg gttcacaagc ctagaggata caagggaacg 660

tccgccaact ttcctttacc tgcaaatgca ggcacagagg ctgctacgga tactaaatct 720

gcaacactca aatatcatga atggcaagtt ggtctagcac tctcttacag attgaacatg 780

ttagttcctt acattggcgt aaactggtca cgagcaactt ttgatgccga cactatccgc 840

atcgctcaac ctaaattggc ctctgctgtt atgaacttga ccacatggaa cccaaccctt 900

ttaggggaag ccacaatgct tgatacttcc aataaattca gtgacttctt acaaatcgct 960

tcgattcaga tcaacaaaat gaagtctaga aaagcttgcg gtttagctat tggtgcaacg 1020

ttaatcgacg ccgacaaatg gtcaatcact ggtgaagcac gcttaatcaa tgaaagagct 1080

gctcacatga atgctcaatt cagattctag 1110

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Leu Pro Val Gly Asn Pro Ala Glu Pro Ser Leu Leu Ile Asp Gly Thr

1 5 10 15

Met Trp Glu Gly Ala Ser Gly Asp Pro Cys Asp Pro Cys Ser Thr Trp

20 25 30

Cys Asp Ala Ile Ser Ile Arg Ala Gly Tyr Tyr Gly Asp Tyr Val Phe

35 4045

Asp Arg Val Leu Lys Val Asp Val Asn Lys Thr Phe Ser Gly Ile Gly

50 55 60

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

65 70 75 80

Arg Pro Asn Val Ala Tyr Gly Arg His Leu Gln Asp Ser Glu Trp Phe

85 90 95

Thr Asn Ala Ala Phe Leu Ala Leu Asn Ile Trp Asp Arg Phe Asp Ile

100 105 110

Phe Cys Thr Leu Gly Ala Ser Asn Gly Tyr Phe Lys Ala Ser Ser Ala

115 120 125

Ala Phe Asn Leu Val Gly Leu Ile Gly Val Lys Gly Ser Ser Leu Thr

130 135 140

Asn Asp Gln Leu Pro Asn Val Ala Ile Thr Gln Gly Val Val Glu Phe

145 150 155 160

Tyr Thr Asp Thr Thr Phe Ser Trp Ser Val Gly Ala Arg Gly Ala Leu

165 170 175

Trp Glu Cys Gly Cys Ala Thr Leu Gly Ala Glu Phe Gln Tyr Ala Gln

180 185 190

Ser Asn Pro Lys Ile Glu Met Leu Asn Val Ile Ser Ser Pro Ala Gln

195 200 205

Phe Val Val His Lys Pro Arg Gly Tyr Lys Gly Thr Ser Ala Asn Phe

210 215 220

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

225 230 235 240

Ala Thr Leu Lys Tyr His Glu Trp Gln Val Gly Leu Ala Leu Ser Tyr

245 250 255

Arg Leu Asn Met Leu Val Pro Tyr Ile Gly Val Asn Trp Ser Arg Ala

260 265 270

Thr Phe Asp Ala Asp Thr Ile Arg Ile Ala Gln Pro Lys Leu Ala Ser

275 280 285

Ala Val Met Asn Leu Thr Thr Trp Asn Pro Thr Leu Leu Gly Glu Ala

290 295 300

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

305 310 315 320

Ser Ile Gln Ile Asn Lys Met Lys Ser Arg Lys Ala Cys Gly Leu Ala

325 330 335

Ile Gly Ala Thr Leu Ile Asp Ala Asp Lys Trp Ser Ile Thr Gly Glu

340 345 350

Ala Arg Leu Ile Asn Glu Arg Ala Ala His Met Asn Ala Gln Phe Arg

355 360 365

Phe

Claims

1. A combined inactivated vaccine for mycoplasma capricolum pneumonia and Chlamydia psittaci disease comprises active ingredients including inactivated Mycoplasma filiformis subspecies capricolum, inactivated Mycoplasma ovipneumoniae and inactivated Chlamydia psittaci MOMP protein;

the MOMP protein of Chlamydia psittaci is a1) or a2) or a 3):

2. The bivalent inactivated vaccine according to claim 1, characterized in that: in the bivalent inactivated vaccine, the ratio of MOMP proteins of the inactivated mycoplasma filiformis goat subspecies, the inactivated mycoplasma ovipneumoniae and the inactivated chlamydia psittaci is 1.2 x 10⁹ccu：(1.2×10⁸-1.2×10¹⁰)ccu：(1-3)mg。

3. An inactivated vaccine for mycoplasma capricolum pneumonia, the active components of which comprise inactivated mycoplasma filiformis subspecies capricolum and inactivated mycoplasma ovipneumoniae.

4. The vaccine of any one of claims 1 to 3, wherein: the mycoplasma filiform goat subspecies is mycoplasma filiform goat subspecies GT-01, and the preservation number of the mycoplasma filiform goat subspecies in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No. 16504.

5. The vaccine of any one of claims 1 to 3, wherein: the mycoplasma ovipneumoniae is mycoplasma ovipneumoniae SH-01, and the preservation number of the mycoplasma ovipneumoniae is CGMCC No.16503 in the common microorganism center of China Committee for culture Collection of microorganisms.

6. The vaccine of any one of claims 1 to 5, wherein: the vaccine also includes an adjuvant.

7. The method for preparing the inactivated vaccine for mycoplasma capricolum pneumonia of any one of claims 3 to 6, comprising the following steps:

8. A method for preparing a combined inactivated vaccine for mycoplasma capricolum pneumonia and Chlamydia psittaci disease as claimed in claim 1, 2, 4, 5 or 6, comprising the steps of:

(c3) preparing MOMP protein of the Chlamydia psittaci;

9. The mycoplasma filiform goat subspecies GT-01 has a preservation number of CGMCC No.16504 in China general microbiological culture Collection center.

10, S1) or S2):

s1) the use of the MOMP protein of Chlamydia psittaci according to claim 1 and/or the Mycoplasma filiformis goat subspecies GT-01 and/or Mycoplasma ovipneumoniae according to claim 9 in the preparation of a combined inactivated vaccine for mycoplasma capricolumni pneumonia, Chlamydia psittaci disease;

s2) use of the filamentous mycoplasma caprine subspecies GT-01 of claim 9 in the preparation of an inactivated vaccine for mycoplasma caprine pneumonia.