CN106636149B - Parabronema skrjabini serine/threonine protein kitase Pj_STPK gene cloning, prokaryotic expression method and application - Google Patents

Parabronema skrjabini serine/threonine protein kitase Pj_STPK gene cloning, prokaryotic expression method and application Download PDF

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CN106636149B
CN106636149B CN201710001526.5A CN201710001526A CN106636149B CN 106636149 B CN106636149 B CN 106636149B CN 201710001526 A CN201710001526 A CN 201710001526A CN 106636149 B CN106636149 B CN 106636149B
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刘春霞
王文龙
冯陈晨
呼和***
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Abstract

The invention discloses Parabronema skrjabini serine/threonine protein kitase Pj_STPK gene clonings, prokaryotic expression method and application, it is sequenced by Parabronema skrjabini transcript profile, the encoding gene predicted and the nematode database announced compare analysis, find out the peculiar gene of Parabronema skrjabini, peculiar gene Pj_STPK is amplified from Parabronema skrjabini with RT-PCR method, after cloning and sequencing, it is building up in prokaryotic expression carrier pET30a, it converts to E.coli BL21, carry out inducing expression, with the immunogenicity of Western-bloting method verifying recombinant protein rSTPK.After Parabronema skrjabini serine/threonine protein kitase Pj_STPK gene recombinant protein is purified, domestic animal Parabronema skrjabini iELISA detection method is established.

Description

Parabronema skrjabini serine/threonine protein kitase Pj_STPK gene cloning, original Nuclear expression method and application
Technical field
The invention belongs to field of biotechnology, specifically, being related to Parabronema skrjabini serine/threonine protein kitase Pj_STPK gene cloning, prokaryotic expression method and application.
Background technique
Parabronema skrjabini disease parasitizes camel, sheep, ox etc. by Parabronema skrjabini (Parabronema skrjabini) Ruminant abomasum causes, and is infected especially with camel more serious.A large amount of polypide parasitisms can lead to affected animal abomasum and be inflamed, go out Blood, ulcer, serious person cause camel dead, very harmful to feeding hunchbacked industry, are a kind of main parasitic worms for endangering China and supporting hunchbacked industry Disease has brought tremendous economic losses to peasants and herdsmen for a long time.Therefore, the monitoring to Parabronema skrjabini disease from now on and anti- System is the emphasis in the ruminants preventing and treating verminosis such as camel.
Since most camels are all distributed in the backward areas of third world countries in the world, so even to this day, state The inside and outside research report about camel Parabronema skrjabini disease is less.Seriously endanger the Parabronema skrjabini of China camel aquaculture Disease correlative study lag, in the prevention and treatment of camel Parabronema skrjabini disease, presently, there are main problem be the absence of for this The fundamental research of disease, the new method for lacking effective prenatal diagnosis method and prevention and treatment.Suitable for many parasitic nematodes Faecal egg inspection technique is used for the diagnosis of the disease, and recall rate is very low, is difficult to clinical practice.The diagnosis of the disease relies primarily on After death dissect (searching polypide in abomasum), such diagnostic method cannot provide reference for early treatment.It is past in actual production Toward being that expelling parasite is blindly administered in the case where no diagnosis basis, cause also to bring such as poison secondary work while economic loss With, there is drug-resistant worm plant and many drawbacks such as livestock products medicament residue is exceeded.
Currently, the prenatal diagnosis method for urgently studying Parabronema skrjabini disease in production practice, mentions for the anti-system of the disease For scientific basis, keep the anti-system of the disease more targeted, improve effect of control, avoids unnecessary economic input, while to be somebody's turn to do The monitoring of disease provides the monitoring means of science.
Summary of the invention
It is an object of the invention to overcome defect existing in the prior art, Parabronema skrjabini serine/Soviet Union's ammonia is provided Pka acid Pj_STPK gene cloning, prokaryotic expression method and application.This method is sequenced by Parabronema skrjabini transcript profile The encoding gene predicted and the nematode database announced compare analysis, find out the peculiar gene of Parabronema skrjabini and carry out function Can annotation, amplify peculiar gene Pj_STPK from Parabronema skrjabini with RT-PCR method, after cloning and sequencing, be building up to original In nuclear expression carrier pET30a, conversion to E.coli BL21 (DE3) carries out inducing expression, is tested with western-bloting method Demonstrate,prove the immunogenicity of Pj_STPK recombinant protein.
Itself the specific technical proposal is:
Parabronema skrjabini serine/threonine protein kitase Pj_STPK gene, nucleotide sequence such as SEQ ID NO: Shown in 1.
The clone of Parabronema skrjabini serine/threonine protein kitase Pj_STPK gene, prokaryotic expression method, including Following steps:
Step 1, design of primers
It is inserted in upstream primer and downstream primer respectively according to the gene order of Pj_STPK with Oligo6.0 design primer Enter restriction enzyme EcoR I, Xho I restriction enzyme site, the synthesis of primer is completed by Hua Da gene Co., Ltd.
The extraction of step 2, Parabronema skrjabini total serum IgE
Reagent specification, which is extracted, according to TaKaRa RNAiso Plus RNA extracts Parabronema skrjabini total serum IgE, it is specific to walk It is rapid as follows:
1. the grinding and homogenate of sample: being transferred quickly to the Parabronema skrjabini sample saved in liquid nitrogen to use Liquid nitrogen precooler Mortar in, with pestle tissue abrasion, liquid nitrogen is continuously added therebetween, until be ground into powder.Then, it is added into mortar suitable The RNAiso Plus of amount, blows and beats repeatedly, until transparent and homogeneous；Then, homogenate is transferred in centrifuge tube, room temperature (15-30 DEG C) Stand 5min；4 DEG C of centrifugation 5min of 12000g；Careful Aspirate supernatant moves into new centrifuge tube.
2. the extraction of total serum IgE: chloroform (1/5 volume of RNAiso Plus) is added into above-mentioned homogenate lysate, covers tightly Centrifuge tube lid, mixes to emulsifying soln and is creamy white；It is stored at room temperature 5min；4 DEG C of centrifugation 15min of 12000g；It is small from centrifuge The heart take out centrifuge tube (homogenate is divided into three layers at this time, it may be assumed that the colourless supernatant containing RNA, middle white albumin layer and have color Lower layer's organic phase.) Aspirate supernatant is transferred in another new centrifuge tube；0.5-1 times of RNAiso Plus is added into supernatant The isopropanol of volume stands 10min after the centrifuge tube that turns upside down mixes well at room temperature；4 DEG C of centrifugation 10min of 12000g.
The cleaning of 3.RNA precipitating: liquid is carefully discarded supernatant.Be added with 75% ethyl alcohol of RNAiso Plus equivalent, gently on Reverse washing centrifuge tube tube wall down carefully discards supernatant liquid after 4 DEG C of centrifugation 5min of 7500g.
The dissolution of 4.RNA: opening centrifuge tube lid, and drying at room temperature precipitates several minutes；After precipitating is dry, it is added suitable RNase-free water dissolution precipitating.
Step 3, Pj_STPK gene RT-PCR amplification
It is cDNA by template reverse transcription of the Parabronema skrjabini total serum IgE of extraction, then using cDNA as template, is set with above-mentioned The specific primer PCR amplification Pj_STPK gene of meter simultaneously verifies its peculiar property.Pcr amplification reaction condition are as follows: initial denaturation 94℃5min；94 DEG C of 30sec are denaturalized, anneal 57 DEG C of 1min, extends 72 DEG C of 1.5min, 35 circulations；Re-extend 72 DEG C of 10min. After PCR amplification, agarose gel electrophoresis detection is carried out.
Step 4, PCR product recycling
It is operated according to Axygen PCR product QIAquick Gel Extraction Kit specification, recycles Pj_STPK gene PCR product.
The connection of step 5, PCR recovery product and pMD19-T Simple carrier
200 μ L centrifuge tubes are taken to add 1 μ L pMD19-T Simple Vector, 4 μ L PCR recovery products and 5 μ L Solution I, centrifugation mix, 16 DEG C of connections overnight.
The conversion of step 6, recombinant plasmid
It is operated according to Beijing Quanshijin Biotechnology Co., Ltd Trans1-T1 competent cell operation instructions, it will even Object of practicing midwifery converts in Trans1-T1 competent cell, is coated on the solid LB media containing antibiotic (Amp), 37 DEG C of trainings It supports overnight.
Step 7, recombinant clone bacterium PCR identification
Picking positive bacterium colony, is inoculated into overnight incubation in antibiotic LB liquid medium, does PCR amplification with culture Identification.
The double digestion identification of step 8, recombinant clone plasmid
Recombinant plasmid is extracted according to Axygen plasmid extraction kit specification, with restriction enzyme Xho I and EcoR I Double digestion and electrophoresis detection are carried out to recombinant plasmid.
Step 9, recombinant clone bacterium sequencing
It is positive recombinant clone bacterium through PCR identification and the identification of plasmid double digestion, Beijing Huada gene company is sent to carry out Bidirectional sequencing.
The recycling of step 10, target gene and expression vector pET30a
Bacterial strain by correct clone strain is sequenced and containing pET30a plasmid, which is inoculated in respectively in LB culture medium, to be cultivated Night extracts plasmid respectively, carries out double enzymes with I pair of recombination cloned plasmids of restriction enzyme Xho I and EcoR and pET30a plasmid It cuts and electrophoresis detection, glue recycles target gene fragment and pET30a segment.
The connection of step 11, target gene and expression vector pET30a
Take 200 μ L centrifuge tubes that the target fragment of 6 μ L recycling, 2 μ L pET30a expression vector segments, 1 μ L T4 DNA is added Ligase and 1 μ L buffer, 16 DEG C of connections are overnight；Building recombinant expression plasmid (pET30a-Pj_STPK, hereinafter referred to as PETSTPK) as shown in SEQ ID NO:2.
Step 12, recombinant expression plasmid transformed competence colibacillus cell
Experience according to Beijing Quan Shijin (TransGen Biotech) Bioisystech Co., Ltd E.coli BL21 (DE3) The operation of state cell operation instructions is coated on by connection product conversion in E.coli BL21 (DE3) competent cell containing anti- 37 DEG C of overnight incubations on the solid LB media of raw element.
Step 13, the identification for recombinantly expressing bacterium
PCR mirror is carried out to the recombinant expression bacterium E.coli BL21 (pETSTPK) of building, hereinafter referred to as BL21 (pETSTPK) The identification of fixed and double digestion is positive recombinant expression bacterium through PCR identification and the identification of plasmid double digestion, send Beijing Hua Da gene public Department carries out bidirectional sequencing, and specific steps are as described in step 7, step 8 and step 9.
Step 14, the optimization for recombinantly expressing bacterium inducing expression condition
Recombinant expression bacterium inducing expression time, inducer IPTG concentration and cultivation temperature are optimized respectively, determined Best inducing expression condition.
The detection of step 15, recombinant protein expression-form
After determining recombinant protein optimum inductive condition, inducing expression 500mL recombinant bacterium, thalline were collected by centrifugation, and thallus is sunk It after shallow lake washes 3 times with PBS, is precipitated with PBS resuspension, addition lysozyme, room temperature 1h, -20 DEG C of multigelations, ice-bath ultrasonic is broken 15min, 12000g are centrifuged 10min, collect precipitating and supernatant, and albumen sample-loading buffer is added and boils 10min, 12%SDS-PAGE Electrophoresis detection protein expression form.
Step 16, recombinant protein solubilization of inclusion bodies and recombinant protein purification
Recombinant expression bacterium 1L, 12000g is induced to be centrifuged 10min and collect thallus, crack according to the above method by optimum inductive condition Bacterium is recombinantly expressed, lysate precipitating is collected by centrifugation, precipitating 8M urea is dissolved, 12000g low-temperature centrifugation 10min collects supernatant Liquid, according to the raw work Ni in Shanghai after being filtered with 0.45 μm of aperture filter+Column protein purification kit operating procedure purification of recombinant proteins.
Step 17, recombinant protein western blotting detection
The recombination Pj_STPK albumen (hereinafter referred to as rSTPK) being purified into is subjected to SDS-PAGE electrophoresis, it will from plastic plate Protein adhesive takes out, and filter paper, pvdf membrane, protein adhesive, filter paper are placed on clip, puts well clamp in order, and 100V 40min turns Film；Pvdf membrane TBST is taken out after transferring film and washs 4min, is repeated 3~4 times, is shaken envelope with the 5% skimmed milk confining liquid room temperature now matched is micro- Close 2h；It takes out pvdf membrane TBST and washs 4min, repeat 3~4 times, by pvdf membrane in the diluted infection Parabronema skrjabini of 1:2000 Camel serum in 4 DEG C overnight incubation；It takes out pvdf membrane TBST and washes 4min, repeat 3~4 times, by pvdf membrane in horseradish peroxidating Micro- shake of room temperature is incubated for 1h in the rabbit-anti camel polyclonal antibody of object enzyme label；It takes out pvdf membrane TBST and washs 4min, repeat 3~4 Time, it takes a picture after being developed the color with ECL.
Parabronema skrjabini serine/threonine protein kitase Pj_STPK gene of the present invention is in detection domestic animal Si Shi Application during secondary flex insect infection.
Compared with prior art, where advantage of the invention:
The present invention is sequenced by Parabronema skrjabini transcript profile, the encoding gene predicted and the nematode database announced Analysis is compared, the peculiar gene of Parabronema skrjabini is found out, amplifies peculiar gene from Parabronema skrjabini with RT-PCR method Pj_STPK after cloning and sequencing, is building up in prokaryotic expression carrier pET30a, is converted to E.coli BL21 (DE3), is induced Expression, with the immunogenicity of western-bloting method verifying Pj_STPK recombinant protein.Parabronema skrjabini of the present invention Serine/threonine protein kitase Pj_STPK gene and its coded product are in detection domestic animal Parabronema skrjabini course of infection Application, the infection of Parabronema skrjabini can be diagnosed, and this method specificity with higher, sensibility and preferable repeat Property.
Detailed description of the invention
Fig. 1 is the PCR testing result of the peculiar gene Pj_STPK of Parabronema skrjabini in the embodiment of the present invention 1；Wherein, M is DNA Marker DL2000；1 is haemonchus contortus；2 be Parabronema skrjabini；3 be Caenorhabditis elegans；4 be disk tailfiber Worm.
Fig. 2 is the PCR amplification electropherogram of gene Pj_STPK in the embodiment of the present invention 1；Wherein, M is DNA Marker DL2000；1-2 is gene Pj_STPK；3 be blank control.
Fig. 3 is recombinant clone plasmid double digestion qualification result in the embodiment of the present invention 1；Wherein, M is DNA Marker DL2000；1-3 is Pj_STPK gene clone plasmid.
Fig. 4 is recombinant expression plasmid pETSTPK double digestion qualification result in the embodiment of the present invention 1；Wherein, M1 DNA Marker DL2000；M2 is DNA Marker DL10000；1-3 is recombinant expression plasmid pETSTPK.
Fig. 5 is the best inducing expression SDS-PAGE electrophoresis knot of recombinant expression bacterium BL21 (pETSTPK) in the embodiment of the present invention 1 Fruit；Wherein, M is protein marker；1 is BL21 (DE3) empty bacterium；Before 2 is BL21 (pET30) inductions；3 be BL21 (pET30) After induction；4 be expression bacterium before BL21 (pETSTPK) induction；5-6 is BL21 (pETSTPK) at 37 DEG C, and 0.1mMIPTG concentration lures After leading 4h, inducing expression bacterium.
Fig. 6 is the SDS-PAGE electrophoresis detection result of recombinant protein expression-form in the embodiment of the present invention 1；Wherein, M is egg White matter marker；1 is BL21 (pETSTPK) cellular lysate object supernatant；2 precipitate for BL21 (pETSTPK) cellular lysate object.
Fig. 7 is the SDS-PAGE electrophoresis detection result of recombinant protein purification in the embodiment of the present invention 1；Wherein, M1 is albumen Matter marker (10kDa-170kDa)；M2 is protein marker (14.9kDa-97.2kDa)；1-2 is the recombinant protein of purifying rSTPK。
Fig. 8 is recombinant protein His label Western-bloting testing result in the embodiment of the present invention 1；Wherein, M is egg White matter marker；1-2 is purification of recombinant proteins rSTPK.
Fig. 9 is the camel positive serum of purification of recombinant proteins rSTPK and infection Parabronema skrjabini in the embodiment of the present invention 1 Reaction；Wherein, M is protein marker；1-2 is that rSTPKS is reacted with the camel positive serum for infecting Parabronema skrjabini；3 are RSTPK is reacted with the camel negative serum for being uninfected by Parabronema skrjabini.
Specific embodiment
Technical solution of the present invention is described in more detail with specific embodiment with reference to the accompanying drawing.
Embodiment 1
It is sequenced by Parabronema skrjabini transcript profile, the encoding gene predicted and the nematode database announced compare point Analysis, finds out the peculiar gene of Parabronema skrjabini, amplifies peculiar gene Pj_ from Parabronema skrjabini with RT-PCR method STPK after cloning and sequencing, is building up in prokaryotic expression carrier pET30a, is converted to E.coli BL21 (DE3), is carried out induction table It reaches, with the immunogenicity of western-bloting method verifying Pj_STPK recombinant protein.
1.1 experimental methods:
1.1.1 design of primers
It is inserted in upstream primer and downstream primer respectively according to the gene order of Pj_STPK with Oligo6.0 design primer Enter restriction enzyme EcoR I, Xho I restriction enzyme site, the synthesis of primer is completed by Hua Da gene Co., Ltd, primer sequence It is shown in Table 1.
1 Pj_STPK gene primer sequence of table
1.1.2 the extraction of Parabronema skrjabini total serum IgE
The operation of reagent specification is extracted according to TaKaRa RNAiso Plus RNA, concrete operations are the same as above-mentioned Total RNAs extraction Method.
1.1.3Pj_STPK gene RT-PCR is expanded
It is cDNA by template reverse transcription of the Parabronema skrjabini total serum IgE of extraction, then using cDNA as template, is set with above-mentioned The specific primer PCR amplification Pj_STPK gene of meter simultaneously verifies its peculiar property.Pcr amplification reaction condition are as follows: initial denaturation 94℃5min；94 DEG C of 30sec are denaturalized, anneal 57 DEG C of 1min, extends 72 DEG C of 1.5min, 35 circulations；Re-extend 72 DEG C of 10min. After PCR amplification, agarose gel electrophoresis detection is carried out.
1.1.4PCR product recycles
It is operated according to Axygen PCR product QIAquick Gel Extraction Kit specification, recycles Pj_STPK gene PCR product.
1.1.5PCR the connection of recovery product and pMD19-T Simple carrier
200 μ L centrifuge tubes are taken to add 1 μ L pMD19-T Simple Vector, 4 μ L PCR recovery products and 5 μ L Solution I, centrifugation mix, 16 DEG C of connections overnight.
1.1.6 the conversion of recombinant plasmid
It is operated according to Beijing Quan Shijin) Bioisystech Co., Ltd Trans1-T1 competent cell operation instructions, it will even Object of practicing midwifery converts in Trans1-T1 competent cell, is coated on the solid LB media containing antibiotic (Amp), 37 DEG C of trainings It supports overnight.
1.1.7 recombinant clone bacterium PCR is identified
Picking positive bacterium colony is inoculated into overnight incubation in the LB liquid medium containing antibiotic (Amp), is done with culture PCR amplification identification.
1.1.8 the double digestion identification of recombinant clone plasmid
Recombinant plasmid is extracted according to Axygen plasmid extraction kit specification, with restriction enzyme Xho I and EcoR I pair of recombinant plasmid carries out double digestion electrophoresis detection.
1.1.9 recombinant clone bacterium is sequenced
It is positive recombinant clone bacterium through PCR identification and the identification of plasmid double digestion, Beijing Huada gene company is sent to carry out Bidirectional sequencing.
1.1.10 the recycling of target gene and expression vector pET30a
Bacterium by correct clone strain is sequenced and containing pET30a plasmid connects to be inoculated in respectively in LB culture medium and cultivate Night extracts plasmid respectively, carries out double digestion and electrophoresis detection with I pair of recombination cloned plasmids of restriction enzyme XhoI and EcoR, Glue recycles target gene fragment and pET30a segment.
1.1.11 the connection of target gene and expression vector pET30a
Take 200 μ L centrifuge tubes that the target fragment of 6 μ L recycling, 2 μ L pET30a expression vector segments, 1 μ L T4 DNA is added Ligase and 1 μ L buffer, 16 DEG C of connections are overnight；Building recombinant expression plasmid (pET30a-Pj_STPK, hereinafter referred to as pETSTPK)。
1.1.12 recombinant expression plasmid transformed competence colibacillus cell
Experience according to Beijing Quan Shijin (TransGen Biotech) Bioisystech Co., Ltd E.coli BL21 (DE3) The operation of state cell operation instructions is coated on by connection product conversion in E.coli BL21 (DE3) competent cell containing anti- 37 DEG C of overnight incubations on the solid LB media of raw element.
1.1.13 the dientification of bacteria is recombinantly expressed
PCR mirror is carried out to the recombinant expression bacterium E.coli BL21 (pETSTPK) of building, hereinafter referred to as BL21 (pETSTPK) The identification of fixed and double digestion is positive recombinant expression bacterium through PCR identification and the identification of plasmid double digestion, send Beijing Hua Da gene public Department carries out bidirectional sequencing, and specific steps are as described in 1.1.7,1.1.8 and 1.1.9.
1.1.14 the optimization of bacterium inducing expression condition is recombinantly expressed
Recombinant expression bacterium BL21 (pETSTPK) is coated on overnight incubation on solid LB media containing Kan, picking single colonie It is seeded in 6mL fluid nutrient medium containing Kan, 37 DEG C of overnight incubations.LB culture medium of the 20mL containing Kan was inoculated in 1:100 in 2nd day In, OD is worked as in 37 DEG C of 150r/min cultures600IPTG inducer is added when reaching 0.6~1.0 to final concentration 1.0mM, respectively 25 DEG C, 30 DEG C, cultivate 4h under the conditions of 37 DEG C of 180r/min after, respectively take 2mL bacterium solution, 1mL surveys light absorption value OD600, another 1mL is collected by centrifugation The denatured by boiling 10min of albumen sample-loading buffer, 12%SDS-PAGE electrophoresis detection are added in bacterial sediment object for thallus.
Recombinant expression bacterium BL21 (pETSTPK) is coated on overnight incubation on the solid LB media containing Kan, picking single bacterium It falls and is seeded in 6mL fluid nutrient medium containing Kan, 37 DEG C of overnight incubations.20mL was inoculated in containing Kan with the ratio of 1:100 in 2nd day In LB culture medium, OD is worked as in 37 DEG C of 150r/min cultures6001mL bacterium solution is taken to compare when reaching 0.6~1.0 as 0h, remaining training It supports and IPTG inducer is added in object to final concentration 1.0mM, 37 DEG C of 150r/min Fiber differentiations.Respectively 0h, 1h, 2h, 3h, 4h, 6h each time point respectively takes 2mL bacterium solution, and 1mL surveys light absorption value OD600, thalline were collected by centrifugation by another 1mL, and egg is added in bacterial sediment object The white denatured by boiling 10min of sample-loading buffer, 12%SDS-PAGE electrophoresis detection.
Recombinant expression bacterium BL21 (pETSTPK) is coated on overnight incubation on solid LB media containing Kan, picking single colonie It is seeded in fluid nutrient medium of the 6mL containing Kan, 37 DEG C of overnight incubations.20mL was inoculated in containing Kan's with the ratio of 1:100 in 2nd day In LB culture medium, OD is worked as in 37 DEG C of 150r/min cultures600When reaching 0.6~1.0, inducer IPTG is added to final concentration 0.05mM,0.1mM,0.2mM,0.5mM,1.0mM,2.0mM.37 DEG C of 150r/min continue to cultivate 4h, respectively take 2mL bacterium solution, and 1mL is surveyed Light absorption value OD600, thalline were collected by centrifugation by another 1mL, the addition denatured by boiling 10min of albumen sample-loading buffer in bacterial sediment object, and 12% SDS-PAGE electrophoresis detection.
1.1.15 the detection of recombinant protein expression-form
After determining recombinant protein optimum inductive condition, inducing expression 500mL recombinant bacterium, thalline were collected by centrifugation, and thallus is sunk It after shallow lake washs 3 times with PBS, is precipitated with PBS resuspension, addition lysozyme, room temperature 1h, -20 DEG C of multigelations, ice-bath ultrasonic is broken Broken 15min, 12000g are centrifuged 10min, collect precipitating and supernatant, and albumen sample-loading buffer is added and boils 10min, 12%SDS- PAGE electrophoresis detection protein expression form.
1.1.16 recombinant protein solubilization of inclusion bodies and purifying
Recombinant expression bacterium 1L, 12000g is induced to be centrifuged 10min and collect thallus, crack according to the above method by optimum inductive condition Bacterium is recombinantly expressed, lysate precipitating is collected by centrifugation, precipitating 8M urea is dissolved, 12000g low-temperature centrifugation 10min collects supernatant Liquid, according to the raw work Ni in Shanghai after 0.45 μm of aperture filter filtering+Column protein purification kit operating procedure purification of recombinant proteins.
1.1.17 recombinant protein western blotting is detected
The recombinant protein being purified into (rSTPK) is subjected to SDS-PAGE electrophoresis, takes out protein adhesive from plastic plate, Filter paper, pvdf membrane, protein adhesive, filter paper are placed on clip, put well clamp in order, 100V 40min transferring film.It is taken out after transferring film Pvdf membrane TBST washes 4min, repeats 3~4 times, shakes closing 2h with the 5% skimmed milk confining liquid room temperature now matched is micro-.Take out pvdf membrane TBST washs 4min, repeats 3~4 times, by pvdf membrane 4 DEG C in the camel serum of the diluted infection Parabronema skrjabini of 1:3000 It is incubated overnight.Take out pvdf membrane TBST wash 4min, repeat 3~4 times, by pvdf membrane horseradish peroxidase-labeled rabbit-anti Micro- shake of room temperature is incubated for 1h in camel polyclonal antibody.It takes out pvdf membrane TBST and washes 4min, repeat 3~4 times, shone after being developed the color with ECL Phase.
1.2 experimental result
1.2.1Pj_STPK gene specific PCR is detected
The nematode sequence that Parabronema skrjabini serine/threonine protein kitase Pj_STPK gene order and NCBI are announced BLAST comparison is carried out, comparison result shows that the sequence is Parabronema skrjabini characteristic sequences.The amplification of Pj_STPK gene PCR is laggard The detection of 1% agarose gel electrophoresis of row, the result is shown in Figure 1.The result shows that: Pj_STPK gene only expands in Parabronema skrjabini RNA Increase about 579bp band out, and does not amplify Pj_STPK base in haemonchus contortus, Caenorhabditis elegans and filaria volvulus Because of band.
1.2.2 the amplification of target gene
After Pj_STPK gene RT-PCR amplification, is detected with 1% agarose gel electrophoresis, as a result see Fig. 2.As the result is shown: There is the band of about 579bp in Pj_STPK gene, is consistent with expected target gene size.
1.2.3 recombinant clone bacterium PCR is identified
Target fragment is transferred to competent cell after connecting with pMD19-T carrier, and 12~16h is cultivated on solid medium, 3 single colonies of picking are inoculated into LB liquid medium, cultivate 12~16h, then using bacterium solution as template PCR amplifications, and carry out 1% fine jade Sepharose electrophoresis detection.Pj_STPK gene cloning bacterium has amplified about 579bp band, obtains the purpose of expected size Band.
1.2.4 the double digestion identification of recombinant clone plasmid
The bacterial strain being positive through bacterium solution PCR identification extracts plasmid, is carried out with restriction enzyme (EcoR I, Xho I) double Digestion, digestion products electrophoresis detection, is as a result shown in Fig. 3.As a result there are two bands, respectively Pj_STPK gene (0.579kb) With pMD19-T (2.7kb), show that target gene is successively inserted into pMD19-T carrier sequence.
1.2.5 recombinant expression bacterium PCR identification
Target fragment is inserted into transformed competence colibacillus somatic cells after pET30a carrier, is coated on solid medium and cultivates 12 ~16h, 3 single colonies of picking are inoculated into 12~16h of culture in LB liquid medium, using bacterium solution as template PCR amplifications, carry out The detection of 1% agarose gel electrophoresis, amplifies the band of about 579bp, is consistent with expected target gene size.
1.2.6 the double digestion identification of recombinant expression plasmid
The bacterial strain being positive through PCR identification extracts plasmid and carries out double digestion with restriction enzyme (EcoR I, Xho I), Digestion products electrophoresis detection, is as a result shown in Fig. 4.As a result there are two bands, respectively (0.579kb and expression carry Pj_STPK gene Body pET30a (5.4kb), the results showed that target gene is successively inserted into expression vector pET30a.
1.2.7 sequencing result and analysis
The clone bacterium and expression bacterium being positive through PCR identification and double digestion identification carry out bidirectional sequencing, the Pj_ being sequenced It is 100% that the Pj_STPK gene nucleotide series that STPK gene order and transcript profile are sequenced, which compare homology, explanation Pj_STPK gene successful clone successfully constructs recombinant expression carrier into carrier pET30a.
1.2.8 the optimization of bacterium inducing expression condition is recombinantly expressed
Recombinant expression bacterium BL21 (pETSTPK) Multiplying culture of culture is to OD600After reaching 0.6~1.0, respectively to its into Row inducing temperature (25 DEG C, 30 DEG C, 37 DEG C), inducer IPTG concentration (0.05mM, 0.1mM, 0.2mM, 0.6Mm, 1.0mM, 2.0mM) optimized with induction time (0h, 1h, 2h, 3h, 4h, 6h).The result shows that recombinant expression bacterium BL21 (pETSTPK) With empty bacterium BL21 (DE3), do not induce after BL21 (pET30) and induction compared with recombinant bacterium BL21 (pET30), had more at 29kDa One band, it is with expected purpose expression product in the same size.It is 37 that bacterium BL21 (pETSTPK), which is recombinantly expressed, in inducing temperature DEG C, induction time 4h is determined as BL21 (pETSTPK) optimum inductive condition when inducer IPTG induced concentration is 0.1mM, As a result see Fig. 5
1.2.9 the detection of recombinant protein expression-form
After determining recombinant protein optimum inductive condition, inducing expression recombinant bacterium, precipitating that thalline were collected by centrifugation cracks thallus, Precipitating and supernatant is collected by centrifugation, carries out 12%SDS-PAGE electrophoresis, discovery recombinant protein mainly exists in the form of inclusion body In precipitating, Fig. 6 is as a result seen.
1.2.10 the protein purification of recombinant protein
After Pj_STPK DNA recombinant expression bacterium Fiber differentiation, thalline were collected by centrifugation, cracking, the precipitating 8M being collected by centrifugation Urea dissolution, then supernatant is collected by centrifugation, according to the raw work Ni in Shanghai after being filtered with 0.45 μm of aperture filter+Column protein purification steps Pillar purifying protein is crossed, purer recombinant protein has been obtained, has as a result seen Fig. 7.
1.2.11 recombinant protein Western-bloting is detected
Western-Blotting detection, recombinant bacterium BL21 are by antibody of Rabbit Anti-His.tag IgG (pETSTPK) occurs specific band at 29kDa, it was demonstrated that on recombinant protein amalgamation and expression pET30a expression vector His label, is as a result shown in Fig. 8.
Camel serum to determine infection Parabronema skrjabini goes out at 29KDa as antibody test recombinant protein rSTPK Specific band is showed, has shown that with the antibody in the camel serum of infection Parabronema skrjabini specificity can occur for recombinant protein Reaction has preferable immunogenicity, and recombinant protein rSTPK is not reacted with the camel serum for being uninfected by Parabronema skrjabini, As a result see Fig. 9.
Embodiment 2
2.1iELISA experimental method
Parabronema skrjabini serine/threonine protein kitase Pj_STPK gene recombinant protein (rSTPK) is used into 0.05M PH9.6 carbonate buffer solution is diluted to certain concentration, and ELISA Plate is added in every 100 μ L of hole, overnight in 4 DEG C of coatings.It takes out within 2nd day ELISA Plate at room temperature discards liquid in its hole, is washed ELISA Plate 3 times with PBST cleaning solution, each static 5min, washing knot The liquid in ELISA Plate is patted dry after beam on filter paper.Every hole adds the 3%BSA confining liquid of 100 μ L, after 37 DEG C of placement 2h, with washing It washs liquid to wash 3 times, stands 5min every time, the liquid in ELISA Plate hole is patted dry after washing.Serum is diluted to one with dilution It is added to after fixed multiple in ELISA Plate hole, 100 μ L are added in 37 DEG C of incubation 1h in every hole.Liquid in hole is discarded, washes 3 with cleaning solution It is secondary, 5min is stood every time, and the liquid in ELISA Plate hole is patted dry after washing.It is added and marks rabbit by the diluted HRP of proper proportion Anti- camel IgG, every hole are added 100 μ L in 37 DEG C of incubation 45min, are washed 3 times with cleaning solution later, stand 5min, washing knot every time ELISA Plate is patted dry after beam.Every hole adds 100 μ L TMB developing solutions, 37 DEG C of colour developing 10min.After colour developing, every hole adds 100 μ L to terminate Liquid terminates reaction, and OD is read in microplate reader450Value
The foundation of 2.2 Parabronema skrjabini iELISA detection methods
2.2.1 the determination of antigen best peridium concentration and serum optimum dilution degree
According to Checkerboard titration method, purified recombinant protein (rSTPK) is subjected to quantitative dilution with coating buffer, keeps purifying anti- Former every hole (100 hole μ L/) loading total amount is respectively 1 μ g, and 2 μ g, 4 μ g, 8 μ g, 16 μ g, antigen initial concentration is rSTPK (2.53 μ G/ μ L), overnight, next day, (serum that 81 parts of positive serums mix in equal volume was as standard positive using known standard positive for 4 DEG C of coatings Serum), negative serum (serum that 9 parts of negative serums mix in equal volume is as standard female serum) by 1:25,1:50,1:100, 5 gradients of 1:200,1:400 are diluted, and rabbit-anti camel IgG-HRP is diluted by 1:5000, are surveyed according to above-mentioned iELISA program Determine OD450Value.According to OD450The P/N value of every group of value calculating, P/N=experimental group/control group=OD positive serum/OD negative serum, Corresponding antigen diluent degree and serum dilution are optimum dilution degree when P/N value maximum.
2.2.2 the determination of ELIAS secondary antibody optimum dilution degree
After the best diluted concentration of antigen and serum diluting multiple has been determined, rabbit-anti camel IgG-HRP presses 1:2500,1: 5000,1:7500,1:100004 gradient dilutions, every hole add 100 μ L, carry out iELISA test.According to OD450Value calculates each The P/N value of secondary antibody dilution, when P/N value maximum, corresponding secondary antibody dilution was best secondary antibody dilution.
2.2.3 repeated experiment
Choose 2 parts of positive serums and 2 parts of negative serums, and using standard positive serum and standard female serum as to shining into Row iELISA detection, each sample are repeated 3 times, and calculate the average and standard deviation of each sample.
2.2.4 the determination of decision-point
According to the optimum reaction condition of above-mentioned determination, 81 parts of Parabronema skrjabini senses are detected with recombinant protein rSTPK antigen The camel positive serum of dye and 9 parts are uninfected by the camel negative serum of Parabronema skrjabini, and carry out statistical analysis, pass through meter Calculate 9 parts of negative serum OD450The average value of valueWith standard deviation (SD).Recombinant antigen iELISA is determined according to following formula Decision-point:
As sample to be tested OD450Value is judged to the positive when being greater than decision-point, is judged as negative when if being less than decision-point.When to be measured Sample OD450When close with critical value, secondary detection is carried out, when testing result is identical as first time testing result, is determined as doubtful Parabronema skrjabini infection.
2.2.5 sensibility and specificity is tested
The camel positive serum of 81 parts of Parabronema skrjabini infection of part is detected with recombinant protein rSTPK antigen and 9 parts are not felt The camel negative serum of Parabronema skrjabini is contaminated, confirms recombinant antigen iELISA sensibility.
IELISA is carried out with 24 positive serums of the recombinant protein rSTPK antigen to several frequently seen helminth mixed infection Specific detection specifically includes that moniezia, haemonchus contortus, lung filaria, Xia Baite nematode, nematodirus, hair buttock line Worm, trichostrongyle, Oesophagostomum, nose fly maggot.
2.2.6 field experiment
140 parts of camel serum to be checked are subjected to iELISA detection.
2.3 experimental result
2.3.1 the determination of best peridium concentration and best serum dilution
Purified recombinant protein rSTPK antigen is loaded by 5 different concentration, it is known that positive serum and feminine gender Serum presses 5 gradient dilutions, carries out iELISA detection, the OD after developing the color according to iELISA450Positive, negative serum known to value OD450There are notable differences for value, the results are shown in Table 2.Calculating the best peridium concentration of antigen according to P/N value is 2 holes μ g/, and serum is best Dilution is 1:50, the results are shown in Table 3.
2 recombinant protein rSTPK antigen Checkerboard titration OD of table450Value
The P/N value of 3 recombinant protein rSTPK antigen iELISA of table detection
2.3.2 the determination of ELIAS secondary antibody optimum dilution degree
OD is detected according to iELISA450Value calculates P/N value size, determines that ELIAS secondary antibody optimum dilution degree is 1:5000, as a result It is shown in Table 4.
The determination of 4 ELIAS secondary antibody best effort concentration of table
2.3.3 repeated experiment
Selection standard is positive respectively, positive, negative serum known to standard female serum and 2 parts, is carried out with recombinant antigen IELISA detection, each sample repeat detection 3 times.Calculate each serum OD450Average and standard deviation, find out the coefficient of variation, As a result the coefficient of variation is respectively less than 10%, shows recombinant antigen The iELISA detection method of foundation has preferable repeatability, as a result such as table 5.
5 recombinant antigen repeated experiment OD of table450Value
2.3.4 the determination of decision-point
By the way that 9 parts are uninfected by with the camel negative serum of Parabronema skrjabini and the camel of 81 parts of Parabronema skrjabinis infection Positive serum carries out the detection of recombinant antigen iELISA method, the results are shown in Table 6.It, will according to Principle of StatisticsSample Product are judged as positive.According to the OD of 9 parts of negative serums450Value, calculates negative serumStandard deviation (SD)=0.025. therefore, Sample Negative, positive critical value=0.210+3 × 0.023=0.304.The criterion of sample Are as follows: sample OD450When value > 0.304, it is determined as the positive；Sample OD450When value < 0.304, it is determined as feminine gender.
6 Pj_STPK recombinant antigen testing result of table
2.3.5 specificity and sensitivity analysis
According to rSTPK antigen in table 7 to the measurement result of 81 parts of camel positive serums and 9 parts of camel negative serums, according to Sensibility calculation formula obtains the OD that detected 79 parts of serum in 81 parts of positive serums450Value both greater than 0.304, remaining 2 Part serum OD450Value is respectively 0.302 and 0.267, wherein 0.302 is close with critical value, repeat after detection result with before Equally, then it is counted as suspected case.OD450Value is less than critical value for 0.267, is determined as feminine gender.Statistical result shows recombination Albumen rSTPK is 97.5% as diagnostic antigen sensibility.
With the iELISA method of foundation, 24 serum samples of the other helminth mixed infections of common 9 kinds are detected.As a result It has been shown that, 24 parts of serum OD450Value is respectively less than critical value 0.304, is determined as feminine gender.The only serum of Parabronema skrjabini infection It is the positive serum of the positive and other parasitic infections without reaction is handed over, shows that established detection method specificity is good, be shown in Table 7。
7 specificity experiments result of table
2.3.6 field experiment
According to the determining best operating condition of experiment, with recombinant protein rSTPK antigen coat ELISA Plate, to adopting 140 portions of white horses with a black mane Hunchbacked serum carries out iELISA detection, has 122 parts of positive serums, positive rate 85.7% in 140 parts of tested serum.At 20 parts It is determined as in negative serum there are 4 parts of serum to be detected as doubtful Parabronema skrjabini infection.Experimental result is as shown in table 8.
8 field experiment result of table
IELISA detection method established by the present invention can diagnose the infection of Parabronema skrjabini, and this method has preferably It is repeated and higher specificity and sensibility.
The foregoing is only a preferred embodiment of the present invention, the scope of protection of the present invention is not limited to this, it is any ripe It knows those skilled in the art within the technical scope of the present disclosure, the simple of technical solution can be become apparent to Variation or equivalence replacement such as pass through the nucleotide sequence of Parabronema skrjabini serine/threonine protein kitase Pj_STPK gene It replaces, lack or adds several nucleotide and form the nucleotide sequence and its coded product with same function, above-mentioned nucleotide The application of sequence and its coded product in preparation domestic animal Parabronema skrjabini infection detection reagent and Related product, each falls within this In the protection scope of invention.
SEQUENCE LISTING
<110>Agricultural University of the Inner Mongol
<120>Parabronema skrjabini serine/threonine protein kitase Pj_STPK gene cloning, prokaryotic expression side
Method and application
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 579
<212> DNA
<213>artificial sequence
<400> 1
atggttatga ggaacgggtc aggcataaag ataattgatt tcgggtcttc gttcttcaat 60
cgacgtgaaa ggtactttta tattcaatca aggttttaca gggcgccgga agttatactt 120
catcaaccgt acgactcggc aatagacgtg tggtcgttcg catgcatcgc gtacgagctc 180
tttatgggga ggcctctcct ccccgggaaa gataactacg atcagataga aagaataagg 240
cgtcttttta acgacatccc atctacaaag tttcgccttg aatttcctca gccaaatctg 300
tttacaacga gatcggctga ctatttgaca ttcgaggacg taagggaaat gatattgact 360
tcgagcgaaa agacagagga caatgagatg ttctttgatt tcgtagcatc aatattaaag 420
ttccacagtg tggaacgccc taaaccgact gagatactca tgcatccgta tctggcaagg 480
ggagtgaggg agttgggcca tattcaacca tgtgacattg agctcgtcga tagaatatct 540
agagacgtcc atcaagcccc gcctcagggc ataggcgtg 579
<210> 2
<211> 1777
<212> DNA
<213>artificial sequence
<400> 2
atggttatga ggaacgggtc aggcataaag ataattgatt tcgggtcttc gttcttcaat 60
cgacgtgaaa ggtactttta tattcaatca aggttttaca gggcgccgga agttatactt 120
catcaaccgt acgactcggc aatagacgtg tggtcgttcg catgcatcgc gtacgagctc 180
tttatgggga ggcctctcct ccccgggaaa gataactacg atcagataga aagaataagg 240
cgtcttttta acgacatccc atctacaaag tttcgccttg aatttcctca gccaaatctg 300
tttacaacga gatcggctga ctatttgaca ttcgaggacg taagggaaat gatattgact 360
tcgagcgaaa agacagagga caatgagatg ttctttgatt tcgtagcatc aatattaaag 420
ttccacagtg tggaacgccc taaaccgact gagatactca tgcatccgta tctggcaagg 480
ggagtgaggg agttgggcca tattcaacca tgtgacattg agctcgtcga tagaatatct 540
agagacgtcc atcaagcccc gcctcagggc ataggcgtg 579
<210> 3
<211> 26
<212> DNA
<213>artificial sequence
<400> 3
gcggaattca tggttatgag gaacgg 26
<210> 4
<211> 26
<212> DNA
<213>artificial sequence
<400> 4
taactcgagc acgcctatgc cctgag
<210> 5
<211> 193
<212> PRT
<213>artificial sequence
<400> 5
Met Val Met Arg Asn Gly Ser Gly Ile Lys Ile Ile Asp Phe Gly Ser
1 5 10 15
Ser Phe Phe Asn Arg Arg Glu Arg Tyr Phe Tyr Ile Gln Ser Arg Phe
20 25 30
Tyr Arg Ala Pro Glu Val Ile Leu His Gln Pro Tyr Asp Ser Ala Ile
35 40 45
Asp Val Trp Ser Phe Ala Cys Ile Ala Tyr Glu Leu Phe Met Gly Arg
50 55 60
Pro Leu Leu Pro Gly Lys Asp Asn Tyr Asp Gln Ile Glu Arg Ile Arg
65 70 75 80
Arg Leu Phe Asn Asp Ile Pro Ser Thr Lys Phe Arg Leu Glu Phe Pro
85 90 95
Gln Pro Asn Leu Phe Thr Thr Arg Ser Ala Asp Tyr Leu Thr Phe Glu
100 105 110
Asp Val Arg Glu Met Ile Leu Thr Ser Ser Glu Lys Thr Glu Asp Asn
115 120 125
Glu Met Phe Phe Asp Phe Val Ala Ser Ile Leu Lys Phe His Ser Val
130 135 140
Glu Arg Pro Lys Pro Thr Glu Ile Leu Met His Pro Tyr Leu Ala Arg
145 150 155 160
Gly Val Arg Glu Leu Gly His Ile Gln Pro Cys Asp Ile Glu Leu Val
165 170 175
Asp Arg Ile Ser Arg Asp Val His Gln Ala Pro Pro Gln Gly Ile Gly
180 185 190
Val

Claims (3)

1. Parabronema skrjabini serine/threonine protein kitase Pj_STPK gene, which is characterized in that its nucleotide sequence is such as Shown in SEQ ID NO:1；The coding albumen of the Parabronema skrjabini serine/threonine protein kitase Pj_STPK gene, Amino acid sequence is as shown in SEQ ID NO:5；The Parabronema skrjabini serine/threonine protein kitase Pj_STPK gene Specific PCR primers, nucleotide sequence is as shown in SEQ ID NO:3, SEQ ID NO:4.
2. the coding albumen of Parabronema skrjabini serine/threonine protein kitase Pj_STPK gene described in claim 1 is being made Application in the product of standby detection domestic animal Parabronema skrjabini course of infection.
3. Parabronema skrjabini serine/threonine protein kitase Pj_STPK gene cloning, recombinant expression method, feature exist In, comprising the following steps:
Step 1, design of primers:
Limit is inserted into upstream primer and downstream primer respectively with Oligo6.0 design primer according to the gene order of Pj_STPK The synthesis of property restriction endonuclease EcoR I processed, Xho I restriction enzyme site, primer are completed by Hua Da gene Co., Ltd；
The extraction of step 2, Parabronema skrjabini total serum IgE
Reagent specification is extracted according to TaKaRa RNAiso Plus RNA and extracts Parabronema skrjabini total serum IgE, and specific steps are such as Under:
21) grinding and homogenate of sample: the Parabronema skrjabini sample saved in liquid nitrogen is transferred quickly to Liquid nitrogen precooler In mortar, with pestle tissue abrasion, it is continuously added liquid nitrogen therebetween, until being ground into powder；Then, it is added into mortar appropriate RNAiso Plus, blow and beat repeatedly, until transparent and homogeneous；Then, homogenate is transferred in centrifuge tube, 15-30 DEG C of room temperature standing 5min；4 DEG C of centrifugation 5min of 12000g；Careful Aspirate supernatant moves into new centrifuge tube；
22) extraction of total serum IgE: chloroform being added into above-mentioned homogenate lysate, and 1/5 volume of RNAiso Plus covers tightly centrifugation Pipe lid, mixes to emulsifying soln and is creamy white；It is stored at room temperature 5min；4 DEG C of centrifugation 15min of 12000g；It is carefully taken from centrifuge Centrifuge tube Aspirate supernatant is transferred in another new centrifuge tube out；0.5-1 times of RNAiso Plus volume is added into supernatant Isopropanol stands 10min after the centrifuge tube that turns upside down mixes well at room temperature；4 DEG C of centrifugation 10min of 12000g；
23) cleaning of RNA precipitate: liquid is carefully discarded supernatant；75% ethyl alcohol with RNAiso Plus equivalent is added, gently up and down Washing centrifuge tube tube wall is overturned, carefully discards supernatant liquid after 4 DEG C of centrifugation 5min of 7500g；
24) dissolution of RNA: opening centrifuge tube lid, and drying at room temperature precipitates several minutes；After precipitating is dry, suitable RNase- is added Free water dissolution precipitating；
Step 3, Pj_STPK gene RT-PCR amplification
It is cDNA by template reverse transcription of the Parabronema skrjabini total serum IgE of extraction, then using cDNA as template, with above-mentioned design Specific primer PCR amplification Pj_STPK gene simultaneously verifies its peculiar property；Pcr amplification reaction condition are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of 30sec are denaturalized, anneal 57 DEG C of 1min, extends 72 DEG C of 1.5min, 35 circulations；Re-extend 72 DEG C of 10min；PCR expands After increasing, agarose gel electrophoresis detection is carried out；
Step 4, PCR product recycling
It is operated according to Axygen PCR product QIAquick Gel Extraction Kit specification, recycles Pj_STPK gene PCR product；
The connection of step 5, PCR recovery product and pMD19-T Simple carrier
200 μ L centrifuge tubes are taken to add 1 μ L pMD19-T Simple Vector, 4 μ L PCR recovery products and 5 μ L Solution I, Centrifugation mixes, 16 DEG C of connections overnight；
The conversion of step 6, recombinant plasmid
It is operated according to Beijing Quanshijin Biotechnology Co., Ltd Trans1-T1 competent cell operation instructions, connection is produced Object converts in Trans1-T1 competent cell, is coated on the solid LB media of the Amp containing antibiotic, 37 DEG C of overnight incubations；
Step 7, recombinant clone bacterium PCR identification
Picking positive bacterium colony, is inoculated into overnight incubation in antibiotic LB liquid medium, makees PCR amplification mirror with culture It is fixed；
The double digestion identification of step 8, recombinant clone plasmid
Recombinant plasmid is extracted according to Axygen plasmid extraction kit specification, it is right with restriction enzyme Xho I and EcoR I Recombinant plasmid carries out double digestion and electrophoresis detection；
Step 9, recombinant clone bacterium sequencing
It is positive recombinant clone bacterium through PCR identification and the identification of plasmid double digestion, send Beijing Huada gene company to carry out two-way Sequencing；
The recycling of step 10, target gene and expression vector pET30a
Bacterial strain by correct clone strain is sequenced and containing pET30a plasmid is inoculated in overnight incubation in LB culture medium respectively, point Indescribably take plasmid, with I pair of recombination cloned plasmids of restriction enzyme Xho I and EcoR and pET30a plasmid carry out double digestion and Electrophoresis detection, glue recycle target gene fragment and pET30a segment；
The connection of step 11, target gene and expression vector pET30a
Take 200 μ L centrifuge tubes that the target fragment of 6 μ L recycling, 2 μ L pET30a expression vector segments, 1 μ L T4 DNA connection is added Enzyme and 1 μ L buffer, 16 DEG C of connections are overnight；Recombinant expression plasmid pETSTPK is constructed as shown in SEQ ID NO:2；
Step 12, recombinant expression plasmid transformed competence colibacillus cell
It operates, will connect according to Beijing Quanshijin Biotechnology Co., Ltd's E.coli BL21 competent cell operation instructions Product converts in E.coli BL21 competent cell, is coated on 37 DEG C of overnight incubations on antibiotic solid LB media；
Step 13, the identification for recombinantly expressing bacterium
PCR identification and double digestion identification are carried out to the recombinant expression bacterium E.coli BL21 of building, through PCR identification and the double enzymes of plasmid Cutting identification is positive recombinant expression bacterium, and Beijing Huada gene company is sent to carry out bidirectional sequencing；Specific steps such as step 7, step Rapid 8 and step 9 described in；
Step 14, the optimization for recombinantly expressing bacterium inducing expression condition
Recombinant expression bacterium inducing expression time, inducer IPTG concentration and cultivation temperature are optimized respectively, determined best Inducing expression condition；
The detection of step 15, recombinant protein expression-form
After determining recombinant protein optimum inductive condition, inducing expression 500mL recombinant bacterium, thalline were collected by centrifugation, and bacterial sediment is used It after PBS washes 3 times, is precipitated with PBS resuspension, addition lysozyme, room temperature 1h, -20 DEG C of multigelations, ice-bath ultrasonic is broken 15min, 12000g are centrifuged 10min, collect precipitating and supernatant, and albumen sample-loading buffer is added and boils 10min, 12%SDS-PAGE Electrophoresis detection protein expression form；
Step 16, recombinant protein solubilization of inclusion bodies and recombinant protein purification
Recombinant expression bacterium 1L, 12000g is induced to be centrifuged 10min and collect thallus by optimum inductive condition, cracking recombination according to the above method Bacterium is expressed, lysate precipitating is collected by centrifugation, precipitating 8M urea is dissolved, 12000g low-temperature centrifugation 10min collects supernatant, According to the raw work Ni in Shanghai after being filtered with 0.45 μm of aperture filter+Column protein purification kit operating procedure purification of recombinant proteins；
Step 17, recombinant protein western blotting detection
The recombination Pj_STPK albumen rSTPK being purified into is subjected to SDS-PAGE electrophoresis, takes out protein adhesive from plastic plate, Filter paper, pvdf membrane, protein adhesive, filter paper are placed on clip, put well clamp in order, 100V 40min transferring film；It is taken out after transferring film Pvdf membrane TBST washs 4min, repeats 3~4 times, shakes closing 2h with the 5% skimmed milk confining liquid room temperature now matched is micro-；Take out PVDF Film TBST washs 4min, repeats 3~4 times, by pvdf membrane 4 in the camel serum of the diluted infection Parabronema skrjabini of 1:2000 DEG C be incubated overnight；Take out pvdf membrane TBST and wash 4min, repeat 3~4 times, by pvdf membrane horseradish peroxidase-labeled rabbit-anti Micro- shake of room temperature is incubated for 1h in camel polyclonal antibody；It takes out pvdf membrane TBST and washs 4min, repeat 3~4 times, after being developed the color with ECL Photograph.
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