CN112300276A - Anti-mycoplasma hyopneumoniae hyperimmune serum and preparation method thereof - Google Patents

Abstract

The invention belongs to the technical field of biological serum preparation, and particularly relates to mycoplasma hyopneumoniae-resistant hyperimmune serum, and further discloses a preparation method of the hyperimmune serum. The mycoplasma hyopneumoniae hyperimmune serum is prepared by taking an inactivated vaccine prepared from a mycoplasma hyopneumoniae DJ-166 strain with the preservation number of CGMCC No.4545 as an immunogen and separating serum from an immune rabbit, has good physical properties, can be quickly dissolved after being added with a diluent, and has no bacteria, mold and mycoplasma growth and no exogenous virus pollution; the hyperimmune serum is derived from rabbit bodies, and is used as a standard serum specific antibody which has positive reaction with mycoplasma hyopneumoniae and negative reaction with mycoplasma hyorhinis antigen and mycoplasma hyofloccosum antigen in specificity test; and the mycoplasma hyopneumoniae extract is stored at the temperature of below-40 ℃, has the effective period of 36 months, has the advantages of strong specificity and good stability, and can be used for preparing mycoplasma hyopneumoniae reagents and standard substances.

Description

Anti-mycoplasma hyopneumoniae hyperimmune serum and preparation method thereof

Technical Field

The invention belongs to the technical field of biological serum preparation, and particularly relates to mycoplasma hyopneumoniae-resistant hyperimmune serum, and further discloses a preparation method of the hyperimmune serum.

Background

Mycoplasma hyopneumoniae (MPS) is commonly called swine enzootic pneumonia, also called swine enzootic pneumonia, and is a swine chronic contact respiratory infectious disease with high morbidity and low mortality caused by Mycoplasma hyopneumoniae (Mhy). MPS is widely distributed around the world, and this disease is reported in most swine countries, with a 30% -90% rate of Mhy infection in herds, manifested primarily as coughing and wheezing, and dark red to gray "flesh-like" or "pancreas-like" changes in the lungs. The death rate of the pig generally suffered from the mycoplasma pneumonia is low, and the main harm of the pig is that the growth of the pig is blocked, the feed conversion rate is greatly reduced, the growth and the development are poor for a long time, and the economic benefit of the pig industry is seriously influenced.

At present, the immunization of the mycoplasma hyopneumoniae vaccine is one of the important measures for preventing and controlling MPS, is also the most effective method for controlling the mycoplasma hyopneumoniae, and can effectively reduce the lung loss, improve the feed conversion rate and shorten the marketing time. Generally, after vaccine immunization, the pig immune challenge test is the most effective means in the evaluation method of the efficacy test. However, screening of experimental pigs is difficult, labor cost and feeding cost are high, and experimental time is as long as two months. Therefore, a detection antibody with high purity, strong specificity and good stability is found, an in vitro relative efficacy detection method is used for replacing animal challenge experiments to realize convenient and economic in vitro efficacy detection, and the detection antibody has positive significance for inoculation of mycoplasma hyopneumoniae vaccines.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to provide the mycoplasma hyopneumoniae hyperimmune serum detection antibody which has high purity, strong specificity and good stability, and can be used for in vitro evaluation of mycoplasma hyopneumoniae vaccines;

the second technical problem to be solved by the invention is to provide a preparation method of the mycoplasma hyopneumoniae hyperimmune serum.

In order to solve the technical problem, the anti-mycoplasma hyopneumoniae hyperimmune serum is prepared by taking an inactivated vaccine prepared from a mycoplasma hyopneumoniae DJ-166 strain with the preservation number of CGMCC No.4545 as an immunogen and separating serum from an immune rabbit.

Specifically, the mycoplasma hyopneumoniae DJ-166 strain inactivated vaccine is prepared by inactivating the mycoplasma hyopneumoniae DJ-166 strain and emulsifying with an oil adjuvant.

The invention also discloses a method for preparing the hyperimmune serum, which comprises the following steps:

(1) immunization: taking the inactivated vaccine of the mycoplasma hyopneumoniae DJ-166 strain with the preservation number of CGMCC No.4545 as an immunogen to immunize healthy white rabbits;

(2) blood collection: and (3) detecting the titer of the antibody 14-21 days after the completion of the immunization, wherein when the titer of the agar-agar antibody is more than or equal to 1: when the rabbit is qualified after 16 hours, the rabbit is subjected to blood sampling;

(3) and (3) separating serum: culturing the collected rabbit blood, and collecting serum for later use through solid-liquid separation;

(4) serum purification: centrifuging the collected serum sample and collecting supernatant, purifying by adopting a protein A affinity column, and collecting a purified sample at a target peak position for later use;

(5) freeze-drying: and (3) sterilizing the collected serum sample, and adding a freeze-drying protective agent for freeze-drying treatment to obtain the serum.

Specifically, in the step (1), the immunization step specifically includes: the first immunization is intramuscular injection, and the immunization dose is 3-5 ml; carrying out second immunization 14 days after the first immunization, wherein the immunization dose is 3-5ml, and injecting at two points; a third immunization was carried out 14 days after the second immunization, with an immunization dose of 3-5ml, and two injections were performed.

Specifically, in the step (3), the step of standing at 37 ℃ for 1-2h and the step of standing at 4 ℃ for 4-24 h.

Specifically, in the step (4), in the protein A affinity column purification step, the column is equilibrated with 10-20mmol/L PB solution, and eluted with 100-300mmol/L sodium citrate solution.

Specifically, the step (4) further comprises the step of adding a Tris-NaCl regulating solution with the pH value of 7.5-8.5 into the purified sample.

Specifically, in the step (4), the protein a affinity column purification step specifically includes: diluting the serum with ice-cold 10-20mmol/L PB solution by 5 times for use; then equilibrating 10-20 columns of 2.5ml protein A affinity column with ice-cold 10-20mmol/L PB solution; subsequently 20ml of the diluted sample was bound to the column; after loading, equilibrating 10-20 columns of 2.5ml protein A affinity column with ice-cold 10-20mmol/L PB solution; eluting with 100-300mmol/L sodium citrate solution, collecting 1ml of each component, immediately adding Tris-NaCl with pH of 8.0, and mixing.

Preferably, the method further comprises the step of lyophilizing the hyperimmune serum, namely, collecting a serum sample for sterilization, and adding a lyophilization protection agent for lyophilization treatment to obtain a serum lyophilized product.

Specifically, the freeze-drying protective agent comprises one or a mixture of more of gelatin, sucrose, trehalose, mannose or calf serum albumin;

the addition amount of the freeze-drying protective agent accounts for 10-40wt% of the total amount of the serum freeze-dried product.

The invention relates to a mycoplasma hyopneumoniae hyperimmune serum, which takes an inactivated vaccine prepared from a mycoplasma hyopneumoniae DJ-166 strain with the preservation number of CGMCC No.4545 as an immunogen and is prepared by separating serum from an immune rabbit, has physical properties of a light yellow spongy loose block and a good shape, can be quickly dissolved after being added with a diluent, has no growth of bacteria, mold and mycoplasma, and has no exogenous virus pollution, and most importantly, the hyperimmune serum disclosed by the invention is derived from a rabbit body, is used as a standard serum specific antibody, has a positive reaction with the mycoplasma hyopneumoniae and a negative reaction with a mycoplasma hyopneumoniae antigen and a mycoplasma hyofloccosum antigen in specificity test; and the mycoplasma hyopneumoniae extract is stored at the temperature of below-40 ℃, has the effective period of 36 months, has the advantages of strong specificity and good stability, and can be used for preparing mycoplasma hyopneumoniae reagents and standard substances.

The preparation method of the anti-mycoplasma hyopneumoniae hyperimmune serum provided by the invention comprises the steps of preparing a mycoplasma hyopneumoniae DJ-166 strain into an inactivated vaccine, immunizing a healthy New Zealand white rabbit, collecting blood and separating serum when the titer of an agar-agar antibody is more than or equal to 1:16, purifying the serum by adopting protein A affinity column chromatography, and carrying out freeze-drying and subpackaging. Compared with the traditional centrifugation method, the serum purified by adopting the protein A affinity chromatographic column method has the concentration effect on the serum, the purity and the concentration of the prepared antibody are effectively improved, and the antibody titer is twice of that of the traditional method; non-specific components are effectively removed, cross reaction with porcine-derived related serum and antigen is low, and specificity is stronger; the hyper-immune serum provided by the invention is prepared from rabbit hyper-immune serum, and the freeze-drying protective agent component is added, so that the storage life is prolonged by 16 months compared with that of the traditional method, and the stability is better.

Drawings

In order that the present disclosure may be more readily and clearly understood, the following detailed description of the present disclosure is provided in connection with specific embodiments thereof and the accompanying drawings, in which,

FIG. 1 is an SDS-PAGE electrophoresis of serum samples before and after the purification step; wherein lane 1 is after purification and lane 2 is before purification;

FIG. 2 is a western-blot of purified sera against strain 2771 and strain DJ-166; wherein, lane 1: 27714 mycoplasma hyopneumoniae mycoprotein; lane 2: DJ-166 strain mycoplasma hyopneumoniae mycoprotein.

Detailed Description

The mycoplasma hyopneumoniae DJ-166 strain is separated from the lung of a Shanxi-diseased pig and is obtained by 2 times of subcloning, the immunogenicity of the strain is good, the protection rate of the immune pig after vaccine preparation is over 80 percent, and the vaccine is preserved in the common microorganism center of China Committee for culture Collection of microorganisms; the preservation address is: the Beijing West Lu No. 1 Hospital No. 3 of Chaoyang district, the preservation number is: CGMCC No. 4545.

Example 1 preparation of inactivated vaccine against Mycoplasma hyopneumoniae DJ166 strain

In this example, the mycoplasma hyopneumoniae DJ-166 was inoculated into a modified CH liquid medium for culture, and the CH liquid medium specifically included:

solution A: mixing brain and heart leachate 2.0g, PPLO broth 5.0g, and deionized water 300ml, stirring to dissolve completely, autoclaving at 116 deg.C for 20 min, and cooling;

and B, liquid B: 5.0ml of 10 XHank's liquid, 1.0g of lactalbumin hydrolysate, 5.0g of yeast extract, 0.8g of sodium pyruvate, 3.0g of peptone, 0.1g of sodium thiosulfate, 0.1% phenol red, 400U/ml of penicillin and 545ml of deionized water. Mixing the above components, stirring completely, filtering with 0.22um filter membrane for sterilization, and storing at 4 deg.C:

and C, liquid C: 140ml of healthy horse serum;

and (3) fully and uniformly mixing the solution A, the solution B and the solution C, and obtaining the mycoplasma hyopneumoniae modified CH liquid culture medium by using 1mol/L sodium hydroxide with the pH value of 7.6.

Preparation of an aqueous phase: inoculating 8% -10% Mycoplasma hyopneumoniae DJ-166 seed into the improved CH liquid culture medium,

culturing in a 37 deg.C fermentation tank with 20% oxygen introduction amount for 3-4 days, and collecting bacterial liquid when the culture medium turns yellow and is slightly turbid and pH value is reduced to 6.5-6.8; counting the live bacteria of the obtained bacterial liquid, wherein the titer of the live bacteria is not less than 10¹⁰CCU/ml; concentrating with 50KD membrane to 1/10 of original volume, centrifuging at 4 deg.C at 10000r/min for 60 min, suspending the precipitated bacteria with Tris-NaCl (pH7.2-7.4) buffer solution, centrifuging and washing for 3 times to obtain 1/100 bacteria suspension of original culture volume; adding the concentrated and purified bacterial suspension into 1.0% thimerosal solution to make the final concentration be 0.01%, uniformly mixing them, placing them at 2-8 deg.C and inactivating them for 12 hr. And (4) measuring the protein concentration by using a BCA kit, wherein the protein concentration of 1/100 concentrated bacterial liquid is more than or equal to 4mg/ml, and the hapten is diluted to 300 ug/ml.

Preparing an oil phase: the mineral oil adjuvant is filled into a sterilization bottle, sterilized for 30 minutes at 121 ℃, and cooled for standby. Adding 1 part of oil phase into a shearing machine, slowly adding 1 part of water phase while stirring at a low speed, and stirring at 10000r/min for 5-8 minutes to obtain the milky oil emulsion inactivated vaccine.

Example 2 serum preparation

The preparation method of the anti-mycoplasma hyopneumoniae hyperimmune serum comprises the following steps:

(1) immunization: taking 6 healthy rabbits, and respectively carrying out intramuscular injection on 3.0 ml/rabbit of the mycoplasma hyopneumoniae inactivated vaccine prepared in the example 2; specifically, the first immunization: intramuscular injection of 3 ml; and (3) second immunization: the injection is carried out 14 days after the first immunization, the immunization dose is 3ml, and the injection is carried out in two points; and (3) third immunization: the injection is carried out 14 days after the second immunization, the immunization dose is 3ml, and the injection is carried out at two points;

(2) blood collection: and (3) detecting the titer of the antibody 14-21 days after the completion of the immunization, wherein when the titer of the agar-agar antibody is more than or equal to 1: the titer is qualified after 16 hours, and the white rabbit can be subjected to heart blood sampling;

(3) serum purification: incubating the collected rabbit blood in an incubator at 37 ℃ for 1 hour, standing at 2-8 ℃ for 4 hours, centrifuging for 10 minutes at 3000r/min, separating and collecting serum for later use;

(4) serum purification: centrifuging the collected serum sample at 10000r/min and 4 ℃ for 10 minutes, taking the supernatant, filtering and sterilizing, and then diluting with an ice-cold 20mmol/L PB solution for 5 standby; equilibrating 2.5ml protein A affinity column 10 columns with ice cold 20mmol/L PB solution; subsequently 20ml of the diluted serum sample was column bound; after the sample loading is finished, balancing 10 columns of 2.5ml protein A affinity columns by using ice-cold 20mmol/L PB solution, eluting by using 100mmol/L sodium citrate solution, collecting 1ml of each component, immediately adding 150 mu L of Tris-NaCl adjusting solution with pH8.0 after the collection is finished, and gently mixing uniformly; after completion of the collection, 2.5ml of protein A affinity column was equilibrated with ice-cold 20mmol/L PB solution for 10 columns, and the operation was repeated to purify the serum, and the purified serum was subjected to SDS-PAGE and Western-Blot detection.

In this example, the SDS-PAGE electrophoresis pattern of the serum sample before and after the protein A affinity column chromatography purification is shown in FIG. 1.

The purified serum of the invention, 2771 strain and DJ-166 strain are respectively subjected to western-blot detection, and the detection results are shown in figure 2. As a result, specific bands appear when the purified serum reacts with antigens of 27714 strain and DJ-166 strain of mycoplasma hyopneumoniae, and the result shows that the purified positive serum can still specifically react with the mycoplasma hyopneumoniae.

Example 3 serum Titer assay

Detecting the titer of mycoplasma hyopneumoniae antibodies by adopting an agar diffusion test, preparing 1% agar powder and 8% NaCl from a 1% agarose plate, adding distilled water, heating in a water bath to fully dissolve, and adding into culture dishes with the volume of about 20ml each dish; flatly placing, solidifying and cooling at room temperature, placing the agar plate on a 7-hole pattern printed in advance, and punching according to the accurate position of the pattern by using a puncher, wherein the hole diameter of a central hole is 4mm, the hole diameters of peripheral holes are 3mm, and the hole pitch is 3 mm. diluting the serum antibody by multiple times, dripping standard agar-agar antigen into the central hole, dripping serum of different dilutions to be detected and positive serum into at least one hole into the peripheral hole, and keeping the peripheral hole full without overflow; and (3) covering the agar plate after sample application, then, horizontally placing the plate in a covered wet box, placing the plate in an incubator at 37 ℃ for incubation, and observing and recording results within 8-24 h. The maximum dilution at which the precipitation line occurs was taken as the titer of the serum, and the final measured serum titer results are shown in table 1 below.

TABLE 1 results of serum antibody titer detection before, after and 21 days after immunization

EXAMPLE 4 serum Freeze-dried product

Adopting bovine serum albumin freeze-drying protective agent, uniformly mixing with 5% of sucrose and 75% of purified serum according to the proportion of 20wt%, subpackaging each bottle with 1ml, and obtaining the required serum freeze-dried product by the freeze-drying procedure shown in the following table 2.

TABLE 2 Freeze drying procedure

The general properties of the obtained serum freeze-dried product are detected, including properties, purity test, vacuum degree and moisture test, and the detection results are shown in table 3.

TABLE 3 Positive serum traits and sterility test results

Note: "-" indicates sterile growth; "+" indicates the presence of bacteria.

As can be seen from the results in Table 3, the positive serum prepared by the scheme of the invention is a light yellow spongy loose block which is rapidly dissolved after being added with a diluent and grows aseptically.

Example 5 serum-specific assay

The positive sera prepared in the above example 2 were subjected to agar diffusion test with mycoplasma hyopneumoniae, mycoplasma hyorhinis and mycoplasma hyofloccosum antigens, respectively, to test their specificities, and positive and negative sera controls for mycoplasma hyorhinis and mycoplasma floccosum were set, respectively, with the test results shown in table 4.

TABLE 4 Positive serum titer determination and specificity test results

Example 6 serum stability assay

The freeze-dried positive serum prepared by the scheme is stored at the temperature of minus 40 ℃, and is subjected to property test, sterile test, titer determination and specificity test at 0 month, 3 months, 6 months, 12 months, 18 months, 24 months, 36 months and 40 months respectively, and the test results are shown in table 5.

TABLE 5 shelf life test results for lyophilized positive sera

As can be seen from the results in Table 5, the lyophilized positive serum product prepared by the invention has no change in properties and sterile growth when stored at-40 ℃ for 40 months; the agar-agar titer and the specificity both meet the quality standard requirements of positive serum; in order to ensure the quality of positive serum, the positive serum is preserved at the temperature below 40 ℃ below zero, and the effective period is 36 months.

Example 7 purification protocol selection

In the preparation method of the hyperimmune serum, a protein A affinity chromatography column method is adopted for purifying the serum sample, and the traditional purification method is a separation method of 3000rpm-4000rpm/min and centrifugation for 10-30 min.

Serum samples were purified by protein a affinity column chromatography as in example 1 and by conventional centrifugation under the same other operating conditions, and the differences in titer, purity and concentration of the hyperimmune serum antibody obtained are shown in table 6 below, and the differences in stability are shown in table 7 below.

TABLE 6 Effect of purification mode on hyperimmune serum antibody titer, purity and concentration differences

TABLE 7 Effect of purification mode on product stability

According to the data, compared with the traditional simple centrifugation method, the serum purified by adopting the protein A affinity chromatography column method has the concentration effect on the serum, the purity and the concentration of the prepared antibody are effectively improved, and the titer of the antibody is twice that of the antibody prepared by the traditional method; non-specific components are effectively removed, cross reaction with porcine-derived related serum and antigen is low, and specificity is stronger; the hyper-immune serum provided by the invention is prepared from rabbit hyper-immune serum, and the freeze-drying protective agent component is added, so that the storage life is prolonged by 16 months compared with that of the traditional method, and the stability is better.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

Claims (10)

1. The hyperimmune serum for resisting mycoplasma hyopneumoniae is prepared with inactivated vaccine prepared with mycoplasma hyopneumoniae DJ-166 strain with preservation number of CGMCC No.4545 as immunogen and through separating serum from immunized rabbit.

2. The mycoplasma hyopneumoniae hyperimmune serum according to claim 1, wherein the mycoplasma hyopneumoniae DJ-166 inactivated vaccine is prepared by inactivating the mycoplasma hyopneumoniae DJ-166 and emulsifying with an oil adjuvant.

3. A method for preparing the hyperimmune serum according to claim 1 or 2, comprising the steps of:

(1) immunization: taking the inactivated vaccine of the mycoplasma hyopneumoniae DJ-166 strain with the preservation number of CGMCC No.4545 as an immunogen to immunize healthy white rabbits;

(2) blood collection: and (3) detecting the titer of the antibody 14-21 days after the completion of the immunization, wherein when the titer of the agar-agar antibody is more than or equal to 1: when the rabbit is qualified after 16 hours, the rabbit is subjected to blood sampling;

(3) and (3) separating serum: culturing the collected rabbit blood, and collecting serum for later use through solid-liquid separation;

(4) serum purification: and centrifuging the collected serum sample, collecting supernatant, purifying by adopting a protein A affinity column, and collecting a purified sample at the position of a target peak.

4. The method for preparing hyperimmune serum according to claim 3, wherein the step (1) of immunizing specifically comprises: the first immunization is intramuscular injection, and the immunization dose is 3-5 ml; carrying out second immunization 14 days after the first immunization, wherein the immunization dose is 3-5ml, and injecting at two points; a third immunization was carried out 14 days after the second immunization, with an immunization dose of 3-5ml, and two injections were performed.

5. The method for preparing hyperimmune serum according to claim 4, wherein in step (3), the rabbit blood culture step comprises the steps of standing at 37 ℃ for 1-2h, and standing at 4 ℃ for 4-24 h.

6. The method for preparing hyperimmune serum according to claim 5, wherein in the step (4), the protein A affinity column purification step comprises equilibrating the column with 10-20mmol/L PB solution and eluting with 100-300mmol/L sodium citrate solution.

7. The method for preparing hyperimmune serum according to claim 6, wherein in the step (4), the step (4) further comprises adding a Tris-NaCl solution with pH of 7.5-8.5 to the purified sample.

8. The method for preparing hyperimmune serum according to claim 7, wherein in the step (4), the protein A affinity column purification step specifically comprises: diluting the serum with ice-cold 10-20mmol/L PB solution by 5 times for use; equilibrating 2.5ml protein A affinity column 10-20 columns with ice cold 10-20mmol/L PB solution; subsequently 20ml of the diluted sample was bound to the column; after loading, equilibrating 10-20 columns of 2.5ml protein A affinity column with ice-cold 10-20mmol/L PB solution; eluting with 100-300mmol/L sodium citrate solution, collecting 1ml of each component, immediately adding Tris-NaCl with pH of 8.0, and mixing.

9. The method for preparing hyperimmune serum according to any one of claims 3-8, wherein the method further comprises the step of lyophilizing the hyperimmune serum, i.e., collecting a serum sample for sterilization, and adding a lyoprotectant for lyophilization to obtain a serum lyophilized product.

10. The method for preparing hyperimmune serum according to claim 9, wherein the lyoprotectant comprises one or more of gelatin, sucrose, trehalose, mannose, or bovine serum albumin;

the addition amount of the freeze-drying protective agent accounts for 10-40wt% of the total amount of the serum freeze-dried product.

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