阅读说明：本技术 一种检测羊棘球蚴抗体用抗原及琼脂扩散板的制备方法 (Preparation method of antigen for detecting echinococcus ovis antibody and agar diffusion plate ) 是由 赵扬扬 赖茂林 王盼举 伏刚 冉智光 吕航 古静 殷远滔 杨元礼 于 2020-07-28 设计创作，主要内容包括：本发明公开了一种检测羊棘球蚴抗体用抗原及琼脂扩散板的制备方法,属于生物检测技术领域。抗原的制备方法为：将羊棘球蚴EG95蛋白核酸序列进行酶切连接,构建到质粒载体上；然后转化入大肠杆菌,构建表达菌株,经IPTG诱导表达后破碎得包涵体,经洗涤,裂解,离心,纯化,透析,浓缩,冻干,PBS稀释,即可。琼脂扩散板的制备方法为：向琼脂糖中加入水和PBS,加热,然后加入聚乙二醇6000、MES和氯化钠,冷却,打孔,封底,即可。本发明提供的抗原和琼脂扩散板,具有灵敏度高,特异性强,通用性强的优点,且具有良好生物安全性,易于规模化生产,特别适合于基层兽医人员对羊血清的临床检测,具有广泛的推广应用价值。(The invention discloses an antigen for detecting an echinococcus ovis antibody and a preparation method of an agar diffusion plate, belonging to the technical field of biological detection. The preparation method of the antigen comprises the following steps: carrying out enzyme digestion connection on the echinococcus ovis EG95 protein nucleic acid sequence, and constructing a plasmid vector; then transforming into escherichia coli, constructing an expression strain, carrying out IPTG induced expression, crushing to obtain an inclusion body, washing, cracking, centrifuging, purifying, dialyzing, concentrating, freeze-drying, and diluting with PBS. The preparation method of the agar diffusion plate comprises the following steps: adding water and PBS into agarose, heating, adding polyethylene glycol 6000, MES and sodium chloride, cooling, perforating, and sealing. The antigen and agar diffusion plate provided by the invention has the advantages of high sensitivity, strong specificity and strong universality, has good biological safety, is easy for large-scale production, is particularly suitable for clinical detection of sheep serum by basic veterinarians, and has wide popularization and application values.)

1. A preparation method of an antigen for detecting an echinococcus ovis antibody is characterized by comprising the following steps:

1) amplifying the EG95 gene fragment by PCR to obtain the nucleotide sequence shown as SEQ ID NO: 1;

2) simultaneously carrying out enzyme digestion on the nucleic acid sequence by adopting EcoR I and Xho I, connecting the nucleic acid sequence to a pET32a plasmid vector, and constructing an expression plasmid;

3) transforming the expression plasmid into escherichia coli BL21(DE3), constructing an expression strain, culturing the expression strain, and carrying out IPTG induced expression to obtain a zymogen liquid;

4) carrying out ultrasonic crushing on the zymocyte liquid to obtain an inclusion body;

5) washing the inclusion body, cracking, centrifuging, taking supernatant, and passing through Ni²⁺-NTA affinity chromatography system purification;

6) and dialyzing the purified product, concentrating, freeze-drying and diluting with Phosphate Buffered Saline (PBS).

2. The method for preparing the antigen for detecting the echinococcus ovis antibody according to claim 1, wherein the step 3) comprises the following steps: inoculating a glycerol strain into an LB liquid culture medium containing Amp according to the volume percentage of 2%, and performing shaking culture at 37 ℃ to obtain a primary zymophyte liquid; transferring the primary zymocyte liquid into LB liquid culture medium containing Amp according to the proportion of 2%, oscillating and enlarging culture at 37 ℃, and inducing expression by IPTG with the final concentration of 1 mM.

3. The method for preparing an antigen for detecting an antibody against echinococcus ovis according to claim 1, wherein in the step 5), the washing solution used for washing the inclusion bodies is prepared by: adding water into NaCl and 1M Tris-HCL (Tris (hydroxymethyl) aminomethane) with the pH value of 8.0 to a constant volume;

preparation of the lysate used: adding water into NaCl, urea, imidazole and 1M Tris-HCL with the pH value of 8.0 to a constant volume.

4. The method for preparing an antigen for detecting an antibody against echinococcus ovis according to claim 1, wherein in the step 5), Ni is added²⁺Preparation of elution buffer B used for NTA affinity chromatography system: adding water into NaCl, urea, imidazole and 1M Tris-HCl with the pH value of 8.0 to a constant volume.

5. The method for preparing an antigen for detecting an antibody against echinococcus ovis according to claim 1, wherein in the step 5), Ni is added²⁺Preparation of elution buffer C for NTA affinity chromatography systems: adding water into NaCl, urea, imidazole and 1M Tris-HCl with the pH value of 8.0 to a constant volume.

6. The method for preparing the antigen for detecting the echinococcus ovis antibody according to claim 1, wherein the step 5) comprises the following steps: washing the inclusion body with a washing solution, then adding the inclusion body into an inclusion body lysate, stirring overnight, centrifuging, and taking supernatant fluid, namely inclusion body denatured fluid of the fusion protein; firstly filtering the inclusion body denaturation liquid by a DS0130 affinity chromatography column, and then filtering by a 0.22 mu m filter to obtain a protein crude filtrate; purifying the crude protein filtrate by using Ni2+ -NTA affinity chromatography system.

7. The method for preparing an antigen for detecting an antibody against echinococcus ovis according to claim 1, wherein in step 6), a dialysis bag of 10KD is used, and the dialysis is performed at 4 ℃ for 16h, and the dialyzate is 1mM PBS.

8. The antigen for detecting antibodies against echinococcus ovis prepared by the method according to any one of claims 1 to 7.

9. A method of preparing an agar plate for detecting the antigen according to claim 8, comprising the steps of:

adding deionized water and 0.1mM PBS into agarose, heating until the agarose is completely melted, then adding polyethylene glycol 6000, MES and sodium chloride, stirring until the agarose is completely melted, cooling to 45-60 ℃, pouring into a plate, punching, picking out the agar in the hole, baking the bottom of the plate, and sealing the bottom.

10. The method for preparing an agar diffuser plate according to claim 9, wherein the agarose, water, 0.1mMPBS, polyethylene glycol 6000, MES and sodium chloride are 0.8:90:10:1:1:0.8 in g: mL: g: g.

Technical Field

The invention belongs to the technical field of biological detection, and particularly relates to an antigen for detecting an echinococcus ovis antibody and a preparation method of an agar diffusion plate.

Background

Echinococcosis in sheep is commonly known as echinococcosis (Hydatidosis) and is a human and animal co-morbidity caused by infection of sheep with Echinococcus granulosus (Eg) larvae of Echinococcus granulosus. It is a parasitic disease of both human and livestock which seriously harms health and is caused by that the continuous-period larvae of Echinococcus granulosus parasitize in the liver, lung and other organs of human body, and is one of five parasitic diseases planned and prevented by the Chinese health department. Echinococcosis is mainly prevalent in 350 counties in rural and pastoral regions in western China, about 5000 million threatened population, the average prevalence rate of echinococcosis in population in plateau regions is 1.20%, and the local prevalence rate is up to more than 12%. According to the calculation of agricultural departments, the number of livestock suffering from echinococcosis in China is more than 5000 ten thousand every year, and the direct economic loss caused by the death of the livestock and the waste of viscera is over 30 billion yuan. Therefore, rapid detection of natural infection by echinococcosis is a problem of great concern. The agar diffusion method is an important method for clinical serum detection, is simple, economical and applicable to operation compared with methods such as ELISA and the like, and is particularly suitable for basic veterinary personnel.

Natural antigens (e.g. encapsulating fluid) were earlier used for echinococcosis serum detection. But it has serious biological safety problem, unreliable source and unstable production, so it is difficult to scale up. And Eg95 is a natural oncosphere antigen with the molecular weight of 24.5k Da and has good immunogenicity. Through years of development, the Eg95 recombinant protein vaccine as the only vaccine for controlling echinococcosis at present can be completely used for animal immune prevention, and commercial production is realized. The scale-up of vaccines requires the evaluation of vaccine immunopotency. The Eg95 genetic engineering antigen is safe and reliable, is convenient to produce, has been used for diagnosing echinococcosis ovis ELISA, and can be used for detecting naturally infected serum and immune serum.

However, there are many specific process problems associated with the Eg95 antigen expression purification and agar diffusion methods. For example, inclusion body denaturation is followed by filtration using a 0.22 μm membrane pore size filter. The denatured solution is treated by high-speed centrifugation or the like, and the filter is still very expensive (completely clogged by 0.2mL of the filtrate). The fermentation antigen is not sufficiently concentrated after being purified, contains urea, imidazole and the like, and needs concentration and desalination. But after urea removal, the inhibition of high-concentration urea on bacteria and protease is lost, and the storage life is insufficient. The common agar diffusion plate formula uses single antigen to detect the antigen, the sensitivity of which is not as good as that of the corresponding antigen such as an envelope solution, and the sensitivity needs to be improved.

Disclosure of Invention

In view of the above, the present invention aims to provide an antigen for detecting echinococcus ovis antibodies and a method for preparing an agar diffusion plate.

Through research, the invention adopts the following technical scheme:

1. a preparation method of an antigen for detecting an echinococcus ovis antibody comprises the following steps:

1) amplifying the EG95 gene fragment by PCR to obtain the nucleotide sequence shown as SEQ ID NO: 1;

2) simultaneously carrying out enzyme digestion on the nucleic acid sequence by adopting EcoR I and Xho I, connecting the nucleic acid sequence to a pET32a plasmid vector, and constructing an expression plasmid;

3) transforming the expression plasmid into escherichia coli BL21(DE3), constructing an expression strain, culturing the expression strain, and carrying out IPTG induced expression to obtain a zymogen liquid;

4) carrying out ultrasonic crushing on the zymocyte liquid to obtain an inclusion body;

5) washing the inclusion body, cracking, centrifuging, taking supernatant, and passing through Ni²⁺-NTA affinity chromatography system purification;

6) and dialyzing the purified product, concentrating, freeze-drying and diluting with Phosphate Buffered Saline (PBS).

Preferably, in the step 3), constructing an expression strain specifically comprises: inoculating a glycerol strain into an LB liquid culture medium containing Amp according to the volume percentage of 2%, and performing shaking culture at 37 ℃ to obtain a primary zymophyte liquid; transferring the primary zymocyte liquid into LB liquid culture medium containing Amp according to the proportion of 2%, oscillating and enlarging culture at 37 ℃, and inducing expression by IPTG with the final concentration of 1 mM. Wherein the final concentration is the concentration of IPTG in LB culture medium.

More preferably, in the step 3), constructing an expression strain specifically comprises: inoculating 2% of glycerol strain into 200mL LB liquid culture medium containing Amp (100. mu.g/mL), and performing shaking culture at 37 deg.C at 150r/min until OD600nm reaches about 0.5 to obtain primary zymogen liquid; transferring the primary zymocyte liquid into 5L LB liquid culture medium containing Amp (100. mu.g/mL) according to the proportion of 2%, carrying out shaking amplification culture at the temperature of 37 ℃ at 150r/min, and carrying out induced expression for 6h by IPTG with the final concentration of 1mM when the OD600nm of the culture solution reaches about 0.8.

Preferably, in the step 4), the power of the ultrasonic crushing is 300W, the mode is that the work is carried out for 5s, the pause is 7s, and the ultrasonic time is 40 min.

Preferably, in the step 5), the washing solution for washing the inclusion bodies is prepared: adding water into NaCl and 1M Tris-HCL (Tris (hydroxymethyl) aminomethane) with the pH value of 8.0 to a constant volume;

preparation of the lysate used: adding water into NaCl, urea, imidazole and 1M Tris-HCL with the pH value of 8.0 to a constant volume.

More preferably, in the step 5), the washing solution for washing the inclusion bodies is prepared by the following method: taking 29.22g of NaCl and 20mL of 1M Tris-HCL with the pH value of 8.0, and adding water to a constant volume of 1000 mL;

the preparation method of the lysis solution used for the inclusion body lysis, namely Buffer A, comprises the following steps: taking 29.22g of NaCl, 480.48g of urea, 0.34g of imidazole and 20mL of 1M Tris-HCL with pH8.0, and adding water to a constant volume of 1000 mL.

Preferably, in said step 5), Ni²⁺Preparation of elution buffer B used for NTA affinity chromatography system: adding water into NaCl, urea, imidazole and 1M Tris-HCL with the pH value of 8.0 to a constant volume.

More preferably, in said step 5), Ni²⁺-elution Buffer B used by NTA affinity chromatography system, i.e. Buffer B comprises 3 concentrations, specifically, 10mmol/L imidazole Buffer B is prepared by the following method: taking 29.22g of NaCl, 480.48g of urea, 0.68g of imidazole and 20mL of 1M Tris-HCL with pH8.0, and adding water to a constant volume of 1000 mL;

the preparation method of 20mmol/L imidazole Buffer B comprises the following steps: taking 29.22g of NaCl, 480.48g of urea, 1.36g of imidazole and 20mL of 1M Tris-HCL with pH8.0, and adding water to a constant volume of 1000 mL;

the preparation method of 50mmol/L imidazole Buffer B comprises the following steps: taking 29.22g of NaCl, 480.48g of urea, 3.4g of imidazole and 20mL of 1M Tris-HCL with pH8.0, and adding water to a constant volume of 1000 mL.

Preferably, in said step 5), Ni²⁺Preparation of elution buffer C for NTA affinity chromatography systems: adding water into NaCl, urea, imidazole and 1M Tris-HCL with the pH value of 8.0 to a constant volume.

More preferably, in said step 5), Ni²⁺The elution Buffer C used in the NTA affinity chromatography system, i.e. Buffer C, is prepared by: taking 29.22g of NaCl, 480.48g of urea, 34g of imidazole and 50mL of 1M Tris-HCL with pH8.0, and adding water to a constant volume of 1000 mL.

Preferably, in the step 5), specifically: washing the inclusion body with a washing solution, then adding the inclusion body into an inclusion body lysate, stirring overnight, centrifuging, and taking supernatant fluid, namely inclusion body denatured fluid of the fusion protein; firstly filtering the inclusion body denaturation liquid by a DS0130 affinity chromatography column, and then filtering by a 0.22 mu m filter to obtain a protein crude filtrate; purifying the crude protein filtrate by using Ni2+ -NTA affinity chromatography system.

More preferably, in the step 5), specifically: washing the inclusion body with a washing solution, adding the washed inclusion body into an inclusion body lysate according to the proportion of 1:10(w/v), and magnetically stirring overnight at 4 ℃; then, centrifuging the mixture for 30min at 4 ℃ and 12000r/min, and taking supernatant which is inclusion body denatured liquid of the fusion protein; firstly, carrying out primary filtration on the inclusion body denatured liquid by a DS0130 affinity chromatography column, namely adding the denatured liquid into an empty column of the affinity chromatography column, allowing the denatured liquid to flow out under the action of gravity, collecting the denatured liquid, eluting the denatured liquid by using an elution buffer solution, and filtering by using a 0.22 mu m filter to obtain a crude protein filtrate; purifying the crude protein filtrate by using a Ni2+ -NTA affinity chromatography system, namely taking a filler, filling the filler into a column, washing the column by using deionized water, balancing the column by using Buffer A, loading the crude protein filtrate, and starting to circularly load the protein filtrate when a penetration peak appears; stopping sampling 15min after the ultraviolet absorption peak reaches the maximum value; then, gradient elution is carried out on hybrid protein by using Buffer B respectively containing 10mmol/L, 20mmol/L and 50mmol/L of imidazole, and elution is continued for 20min after the ultraviolet absorption peak reaches the lowest value; and finally, eluting the target protein by using Buffer C, and starting to collect the sample when a Buffer C elution peak appears until the elution peak is completely collected.

Preferably, in the step 6), a 10KD dialysis bag is adopted for dialysis at 4 ℃ for 16h, and the dialysate is 1 mMPBS.

Preferably, in the step 6), the specific operations of dialyzing, concentrating, freeze-drying and diluting with Phosphate Buffered Saline (PBS) are as follows: dialyzing the purified protein with a 10KD dialysis bag, wherein the dialysate is 1mM PBS, dialyzing at 4 ℃ for 16h, and replacing the dialysate every 4h, and precooling the dialysate at 4 ℃; concentrating dialyzed protein with polyethylene glycol PEG20000, lyophilizing by vacuum lyophilization, and packaging 1mL of protein solution in 10mL penicillin bottles, vacuum lyophilizing at-40 deg.C for 48 h; freeze-drying the concentrated dry powder, and preserving at-20 ℃; the dry powder is added with 0.01mM phosphate buffer solution with pH7.4 to be diluted to 1.0mg/mL, and the antigen for detecting the echinococcus ovirinus antibody is obtained.

2. The antigen for detecting the echinococcus ovis antibody prepared by the preparation method.

3. The preparation method of the agar diffusion plate for detecting the antigen comprises the following steps:

adding deionized water and 0.1mM PBS into agarose, heating until the agarose is completely melted, then adding polyethylene glycol 6000, MES and sodium chloride, stirring until the agarose is completely melted, cooling to 45-60 ℃, pouring into a plate, punching, picking out the agar in the hole, baking the bottom of the plate, and sealing the bottom.

Preferably, the agarose, water, 0.1mM PBS, polyethylene glycol 6000, MES and sodium chloride are 0.8:90:10:1:1:0.8 in g: mL: mL: g: g.

The invention has the beneficial effects that:

1) the antigen for detecting the echinococcus ovis antibody provided by the invention has strong universality, can detect natural infection serum as well as immune serum, can detect goats and sheep, has definite antigen components and high purity, and avoids the biosafety problem caused by using natural antigens such as echinococcosis inclusion fluid and the like;

2) the preparation method of the antigen for detecting the echinococcus ovis antibody provided by the invention has the advantages that the production process is stable, safe and reliable, the large-scale production is easy, the denatured liquid is firstly filtered through an affinity chromatography column empty column for rough filtration, and then filtered through a 0.22 mu m filter membrane, the filter membrane blockage is effectively reduced, and the filter membrane cost and the labor cost are saved; the antigen adopts PEG20000 dialysis concentration and freeze-drying treatment process, and has long shelf life;

3) the agar diffusion plate for detecting the antigen provided by the invention adopts agarose as the agar diffusion plate, has good transparency and hardness, is not easy to break and is easy to punch, and PEG6000 and MES are added into the agar diffusion plate, so that the agar diffusion plate has high sensitivity, clear precipitation lines and no occurrence of a plurality of precipitation lines. The detection method is simple to operate, economical and applicable, and suitable for detection of base-level personnel;

4) the antigen and the agar diffusion plate for detecting the echinococcus ovis (hydatid) antibody, provided by the invention, establish a genetic engineering expression antigen production process which has good biological safety and good process stability and is easy for large-scale production in order to realize the detection of natural infection of the echinococcus ovis (hydatid) and EG95 vaccine immune serum, and establish an agar diffusion method which has high sensitivity, strong specificity and strong universality by utilizing the expression antigen, so that the method has the advantages of economy and simplicity, is particularly suitable for clinical detection of the goat serum by basic veterinarians, and has wide popularization and application values.

Drawings

FIG. 1 is a diagram showing the identification of PCR amplification in example 1 of the present invention;

FIG. 2 is a diagram showing the identification of enzyme digestion in example 1 of the present invention;

FIG. 3 is a graph showing the expression of a target protein before and after induction by bacteria in example 1 of the present invention;

FIG. 4 is a SDS-PAGE analysis result of the protein before and after purification in example 1 of the present invention;

FIG. 5 is a Western-Blot analysis result of the antigen for detecting echinococcus ovis antibody prepared in example 1 of the present invention;

FIG. 6 is a graph showing the effect of different agarose ratios on the precipitation line in example 3 of the present invention;

FIG. 7 is a graph showing the comparison of AGP detection response times in example 3 of the present invention;

FIG. 8 is a graph showing the results of the dilution of antigens for detecting echinococcus ovis antibodies in example 3 of the present invention;

FIG. 9 shows the degree of the test results of the dilution of the echinococcus EG95 antibody positive serum in example 3 of the present invention;

FIG. 10 is a diagram showing the specificity of the AGP detection method in example 3 of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.