CN109879959A - The method of prokaryotic expression and its polyclonal antibody preparation of giant panda IgG Fc segment - Google Patents

Abstract

The invention discloses methods prepared by the prokaryotic expression of giant panda IgG Fc segment and its polyclonal antibody, prokaryotic expression giant panda IgG Fc constant region is selected for the first time, DH5 α bacterium is converted with the recombinant vector pMAL-C4x-PFc containing giant panda IgG Fc fragment gene built, it extracts after plasmid is identified with BamH I and Hind III digestion and converts BL21 bacterium, by the optimization of inductive condition, the great amount of soluble expression of giant panda IgG Fc segment is realized in success, with the isolated purer recombinant protein of affinity chromatography；3 immune health mouse after the purer soluble recombinant protein and Freund's adjuvant that affinity chromatography is obtained are fully emulsified, mouse serum is acquired after 3 times immune 1 week, it is very high with indirect ELISA survey polyclonal antibody potency, it is successfully prepared the giant panda polyclonal antibody that potency is high, stability is good.

Description

The method of prokaryotic expression and its polyclonal antibody preparation of giant panda IgG Fc segment

Technical field

The invention belongs in the field of biotechnology, and in particular to a kind of prokaryotic expression of giant panda IgG Fc segment and its The method of polyclonal antibody preparation.

Background technique

Currently, giant panda is rare animal in imminent danger, our countries take many measures to save and protect giant panda, For example giant panda nature reserve area is established, population quantity etc. is increased by artificial breeding giant panda.Its battalion of Take care of pandas It is more much higher than field to support configuration, but the service life is significantly lower than the giant panda of field survivorship, and Captive Giant Panda fertility is bright Aobvious decline；In addition, epidemic disease problem is also to perplex one of the critical bottleneck problem of Panda protection under barrier conditions.Effective disease Disease detection is significant for protection giant panda, but the method for detecting specificity of current diagnosis giant panda epidemic disease is less, this with lack Few giant panda specificity antiantibody is related.

More than half exists the recombinant protein of the prokaryotic expression system inducing expression of Escherichia coli in the form of inclusion body, can Dissolubility albumen is only the 13.3%-23% that summary table reaches albumen, can not great amount of soluble expression albumen.

In view of this, the present invention is specifically proposed.

Summary of the invention

The object of the present invention is to provide method prepared by the prokaryotic expression of giant panda IgG Fc segment and its polyclonal antibody, Prokaryotic expression giant panda IgG Fc constant region is selected for the first time, contains giant panda IgG Fc fragment gene with what is built Recombinant vector pMAL-C4x-PFc convert DH5 α bacterium, extract plasmid with BamH I and Hind III digestion identify after convert BL21 Bacterium successfully realizes the great amount of soluble expression of giant panda IgG Fc segment, uses Dextrin by the optimization of inductive condition The isolated purer recombinant protein of Beads 6FF affinity chromatography；The purer soluble recombinant protein that affinity chromatography is obtained With Freund's adjuvant it is fully emulsified after 3 immune health mouse, acquire mouse serum after 3 times immune 1 week, survey more grams with indirect ELISA Grand antibody titer is very high；Meanwhile with the polyclonal antibody of preparation and giant panda serum IgG reaction, reactivity is good, 3 weights Retrial is tested, and testing result consistency is good；With health pig, sheep, rabbit and chicken blood clearance response, the antiantibody of preparation and pig, sheep There are cross reactivities with rabbit serum, faint with chicken serum cross reaction.It is good that this success of the test is prepared for potency height, stability Giant panda polyclonal antibody, the polyclonal antibody of preparation is coupled in conjunction with enzyme and forms enzyme-labelled antigen, it is more to can be used for giant panda The serodiagnosis of kind infectious disease, parasitic disease and other diseases；Meanwhile it can be marked with horseradish peroxidase (HRP) Mountain sheep anti-mouse igg ELIAS secondary antibody is established indirect ELSIA method and is analyzed for giant panda serum antibody, tight especially for certain The important epidemic disease of giant panda health is threatened again.

To achieve the goals above, the side of the prokaryotic expression of a kind of giant panda IgG Fc segment (PFc) provided by the invention Method, comprising the following steps:

(1) construct recombinant expression carrier: by PFc insertion pMAL-C4x carrier BamH I and Hind III digestion site it Between, obtain pMAL-C4x-PFc recombinant vector；

(2) recombinant plasmid transformed bacillus coli DH 5 alpha bacterium, by pMAL-C4x-PFc recombinant vector obtained in step (1) with The DH5 α bacterium of culture is mixed, and the recombination DH5 α bacterium of expression PFc is obtained；

(3) extraction of recombinant plasmid: recombinating DH5 α bacterium obtained in ammonia benzyl resistance screening step (2), cultivates ammonia benzyl resistance Recombination DH5 α bacterium to OD600 value after screening reaches 0.6-0.8, the recombination of the recombination DH5 α bacterium after culture is extracted using kit Plasmid obtains pMAL-C4x-PFc recombinant plasmid；

(4) recombinant plasmid transformed glycerol stock BL21 bacterium: by pMAL-C4x-PFc recombinant plasmid obtained in step (3) and training Feeding BL21 bacterium is mixed, and recombination BL21 bacterium is obtained；

(5) it recombinates the inducing expression of BL21 bacterium: recombinating BL21 bacterium obtained in ammonia benzyl resistance screening step (4) and carry out Culture to OD600 value reaches 0.6-0.8, and using the recombination BL21 bacterium after inducer inducing expression culture, albumen is used after centrifugation Purifying combines equilibration buffer that precipitating is resuspended, and the recombination BL21 bacterium thallus after ultrasonication resuspension is obtained to bacteria liquid has translucency To the recombinant protein of expression.

Further, further include the identification of recombinant plasmid in step (3): measurement recombinates the dense of the recombinant plasmid of DH5 α bacterium The concentration of the recombinant plasmid of degree and original pMAL-C4x-PFc recombinant vector, using two kinds of restriction nucleases of BamH I and Hind III The recombinant plasmid of restriction endonuclease cutting recombination DH5 α bacterium and the recombinant plasmid of original pMAL-C4x-PFc recombinant vector, and measure cutting The plasmid concentration of each recombinant plasmid afterwards；And the weight of the recombinant plasmid and original pMAL-C4x-PFc recombinant vector of DH5 α bacterium will be recombinated Group plasmid and the digestion products of their BamH I and Hind two kinds of restriction endonuclease of III cutting use nov nucleic acid Swimming carries out size comparison.

Preferably, in step (5), using the recombination BL21 bacterium after inducer inducing expression culture, wherein inducing temperature is 25-40℃。

Preferably, in step (5), using the recombination BL21 bacterium after inducer inducing expression culture, wherein inducer is IPTG, inducer concentrations 0.1-1.5mmol/L.

Preferably, in step (5), using the recombination BL21 bacterium after inducer inducing expression culture, wherein induction time is 1-8h。

Preferably, in step (5), ultrasonication be resuspended after recombination BL21 bacterium thallus when, surpassed in ice bath Sound, and the every ultrasound interval 4-6s 2-4s is abundant to ensure to radiate.

Further, further include step (6): the purifying of recombinant protein: using and obtained in affinity chromatography purification step (5) Recombinant protein, wherein in eluent for purifying recombinant protein.

It is obtained the present invention also provides a kind of method of prokaryotic expression using above-mentioned giant panda IgG Fc segment (PFc) The method that recombinant protein prepares polyclonal antibody, comprising the following steps:

S1: the measurement of recombinant protein concentration: the giant panda IgG Fc segment of the isolated purifying of affinity chromatography is used Prokaryotic expression recombinant protein, using protein gray analysis software survey purifying recombinant protein IOD value, use albumen Marker obtains the concentration value of the recombinant protein of purifying as reference；

S2: the preparation of polyclonal antibody: by the recombinant protein of purifying and equivalent Freund's complete adjuvant mixing and emulsifying, emulsification is extremely It instills 15s in water not scatter, the recombinant protein branch subcutaneous injection mouse after emulsification is immunized, every two weeks once, altogether It is immunized three times, wherein the injection volume of the preceding injection of mouse twice recombinant protein reaches 80-120 μ g, 6-8 after third time is immune It, carries out eyeball blood sampling to immune mouse, and blood is placed at room temperature for after its natural coagulation, is carried out centrifuging and taking supernatant, that is, serum, is obtained To polyclonal antibody.

Further, further include step S3: the detection of polyclonal antibody potency: being marked using horseradish peroxidase (HRP) Remember mountain sheep anti-mouse igg ELIAS secondary antibody, it is dense to determine that recombinant protein is most preferably coated with dilution using enzyme-linked immunosorbent assay (ELISA) Degree, purification of recombinant proteins after being diluted to best peridium concentration dilution ratio after treatment respectively with immune mouse, nonimmune Doubling dilution is carried out after the serum mixing of mouse, addition horseradish peroxidase (HRP) label goat is anti-after being incubated for washing drying Mouse IgG ELIAS secondary antibody is incubated for, and the potency of polyclonal antibody is calculated using indirect enzyme-linked immunosorbent assay (ELISA).

The present invention also provides a kind of giant panda prepared using the above-mentioned method for preparing polyclonal antibody is polyclonal Antibody.

The method of prokaryotic expression and its polyclonal antibody preparation of giant panda IgG Fc segment provided by the invention, has such as It is lower the utility model has the advantages that

1, prokaryotic expression giant panda IgG Fc constant region is selected, MBP fusion tag is selected to promote giant panda IgG The great amount of soluble of Fc segment is expressed, and the recombination large intestine that can express giant panda IgG Fc segment is obtained using chemical transformation Bacillus, while convenient for utilizing the isolated purer recombinant protein of affinity chromatography；Contain giant panda IgG Fc for what is built The recombinant vector pMAL-C4x-PFc of fragment gene converts BL21 bacterium, has determined by the optimization of inductive condition containing giant panda The relatively suitable condition of the recombinant bacterium expression of IgG Fc segment is 37 DEG C, and IPTG concentration 0.5mM, inducing expression 5h are successfully realized The great amount of soluble of giant panda IgG Fc segment is expressed；It is successfully obtained using Dextrin Beads 6FF affinity protein purification Purer soluble recombinant protein, the mean concentration of soluble recombinant protein reach 265.85 μ g/mL；

2, detected with polyclonal antibody potency, stability and specificity of the ELISA method to preparation, preparation it is more Clonal antibody and giant panda serum IgG reaction are good, and test variance rate is lower, and polyclonal antibody potency is high, stability is preferable； There are cross reactivities for the polyclonal antibody and pig, sheep and rabbit serum of preparation, faint with chicken serum cross reaction.

Detailed description of the invention

Fig. 1 is the electrophoretogram of the double digestion method identification pMAL-C4x-PFc recombinant plasmid in specific embodiment.

Fig. 2 is the electricity of the expression of results of the pMAL-C4x-PFc recombinant plasmid in specific embodiment under different inducing temperatures Swimming figure.

Fig. 3 is the expression knot of the pMAL-C4x-PFc recombinant plasmid in specific embodiment under IPTG inducer various concentration The electrophoretogram of fruit.

Fig. 4 is the table of the pMAL-C4x-PFc recombinant plasmid in specific embodiment under IPTG inducer difference induction time Up to the electrophoretogram of result.

Fig. 5 is the electricity of the expression of results of pMAL-C4x-PFc recombinant protein under suitable expression condition in specific embodiment Swimming figure.

Fig. 6 is the electrophoretogram of the purification result of pMAL-C4x-PFc recombinant protein in specific embodiment.

Fig. 7 is the source of mouse polyclonal antibody and giant panda serum, pig, sheep, rabbit and chicken serum prepared in specific embodiment The inspection result of atopic.

Specific embodiment

In order to enable those skilled in the art to better understand the solution of the present invention, With reference to embodiment to this hair It is bright to be described in further detail.

One, the prokaryotic expression of giant panda IgG Fc segment and purifying

1, material

(1) plasmid and cell

The bacillus coli DH 5 alpha and e. coli bl21 saved at a temperature of -80 DEG C of this laboratory, pMAL-C4x-PFc recombination Carrier is constructed by company's early period and is saved.

(2) main solution

DNA sample-loading buffer, 1 × TAE solution, TSB culture medium, ammonia benzyl resistance LB (Luria-Bertani) culture medium, nothing Resistance LB (Luria-Bertani) culture medium, 2YT culture medium, KCM solution, SDS-PAGE electrophoretic buffer, 75% glycerol are molten Liquid, SDS-PAGE sample-loading buffer, 10% ammonium persulfate, coomassie brilliant blue R_250 dyeing liquor and destainer, MBP label protein Purifying combination/wash miscellaneous buffer and elution buffer, IPTG (isopropylthiogalactoside) solution.

(3) key instrument equipment

Pure water meter, high-pressure steam sterilizing pan, nucleic acid electrophoresis apparatus, micro-spectrophotometer, superclean bench, water-bath, perseverance Temperature oscillation shaking table, gel imaging system, centrifuge, electronic balance, micro-wave oven, refrigerator, constant incubator, liquid-transfering gun, albumen electricity Swimming instrument, ice machine, Ultrasonic Cell Disruptor.

2, the method for the prokaryotic expression of giant panda IgG Fc segment and purifying, comprising the following steps:

(1) building of recombinant expression carrier

The giant panda IgG Fc segment (PFc) included according to GenBank, by the BamH I of PFc insertion pMAL-C4x carrier Between Hind III digestion site, pMAL-C4x-PFc recombinant vector is synthesized.

(2) recombinant vector converts DH5 α bacterium

The DH5 α bacterium that -80 DEG C are saved takes out dissolution, carries out scribing line recovery on the LB solid culture ware of non-resistant, crosses LB solid culture ware afterwards is inverted in 37 DEG C of constant temperature incubators and is incubated overnight 12-16h.It is trained after culture with white pipette tips picking LB solid The single bacterium grown on ware is supported to fall in 3mL non-resistant LB liquid medium in 37 DEG C of 200rpm constant temperature oscillation shaker overnight cultures 12~16h, bacterium solution is fog-like can stop cultivating for oscillation observation.3mL bacterium solution after culture is poured into the micro of 1.5mL in two times Centrifuge tube, is centrifuged 1min twice with 12000rpm respectively, is centrifuged all discards supernatant solution twice, retains precipitating.To twice from The TSB culture medium that 50 μ L are added in precipitating after the heart sufficiently suspends precipitating, add the pMAL-C4x-PFc recombinant vector of 3 μ L with The KCM solution of 10 μ L mixes well, and in addition takes the 100 μ L system of non-resistant LB liquid medium polishing of 37 μ L, after ice bath 20min It is placed at room temperature for 10min, in the non-resistant LB liquid medium that 500 μ L are added in super-clean bench into above-mentioned microcentrifugal tube, in 37 15min is cultivated on DEG C 200rpm constant temperature oscillation shaking table, 5000rpm is centrifuged 5min later, discards supernatant solution, retains precipitating, uses The non-resistant LB liquid medium of 200 μ L is resuspended, and draws the LB solid culture that 100 μ L bacterium solutions are coated with ammonia benzyl resistance with tripod Ware is inverted in 37 DEG C of constant temperature incubators and is incubated overnight, and observes bacterium colony growing state, remaining 100 μ L bacterium solutions be put in 4 DEG C it is spare.

(3) extraction and identification of recombinant plasmid

The LB liquid training of the ammonia benzyl resistance of 5mL is fallen with the DH5 α single bacterium on white pipette tips picking in step after ammonia benzyl resistance screening It supports in base in 37 DEG C of 200rpm constant temperature oscillation shaker overnight culture about 14h, is extracted according to the small extraction reagent kit specification of plasmid 50 μ L of plasmid.The plasmid concentration extracted is measured with micro-spectrophotometer and uses two kinds of BamH I and Hind III restricted The plasmid concentration of endonuclease cut and extract, the pMAL-C4x-PFc saved originally with micro-spectrophotometer measurement experiment room The concentration of recombinant plasmid and the pMAL-C4x- for using two kinds of restriction endonuclease cut and extracts of BamH I and Hind III The concentration of PFc recombinant plasmid, digestion condition are 37 DEG C of water-bath 4h, and the specific ingredient of digestion system is shown in Table 1.

1 endonuclease reaction system of table (unit: μ L)

1 endonuclease reaction system of table (unit: μ L)

1 × TAE solution that 25mL is added in 0.25g agarose is weighed, microwave stove heating 2min takes out every 1min and rocks It sees whether to dissolve, be taken out after the transparent liquid that becomes colorless after it.When temperature is down to 50~60 DEG C, 2.5 μ are added thereto The Goldview of L is mixed, and wherein GoldView is the new nucleic acid dyestuff that one kind can replace ethidium bromide (EB), is poured into slotting In the groove of good comb, 2~4h or so is stood.Solidified 1% agarose nucleic acid gel is put into nucleic acid electrophoresis apparatus, it is former Recombinant plasmid, the positive bacterium colony plasmid of extraction and their digestion products are sufficiently mixed rear point sample with DNA sample-loading buffer respectively, Whether plasmid is extracted in nucleic acid electrophoresis verifying correct.The qualification result of recombinant plasmid is shown in Fig. 1.

As shown in Figure 1, M is classified as DNA Marker in figure, A is classified as former recombinant plasmid in figure, and B is classified as former recombinant plasmid in figure Double enzyme digestion product, C is classified as the positive bacterium colony plasmid of extraction in figure, and D is classified as the positive bacterium colony plasmid double enzyme digestion product of extraction in figure；Recombination Plasmid pMAL-C4x-PFc conversion DH5 α bacterium ammonia benzyl resistance screening goes out positive bacteria and falls behind successfully to extract plasmid, with micro spectrophotometric The recombinant plasmid concentration for measuring to extract positive bacterium colony is 220.6ng/ μ L.With two kinds of restriction nucleases of BamH I and Hind III The pMAL-C4x-PFc recombinant plasmid that the plasmid of restriction endonuclease cut and extract positive bacterium colony and laboratory saved originally, by original weight group Plasmid, the positive bacterium colony plasmid of extraction and their digestion products and DNA Marker run nucleic acid electrophoresis and are compared, and discovery is former Recombinant plasmid and the positive bacterium colony plasmid of extraction are in the same size, their digestion products size is also consistent.And giant panda IgG Fc piece Section mrna length is 1035bp, correct according to DNA Marker comparison digestion products, obtains correct pMAL-C4x-PFc Recombinant plasmid.

(4) recombinant plasmid transformed BL21 bacterium

The BL21 bacterium that -80 DEG C are saved takes out room-temperature dissolution, recovery of crossing on the LB solid culture ware of non-resistant, in 37 It is inverted in DEG C constant temperature incubator and is incubated overnight 12-16h.With growing on the LB solid culture ware of white pipette tips picking non-resistant after culture Single bacterium falls within 3mL non-resistant LB liquid medium and is incubated overnight 12~16h, oscillation observation in 37 DEG C of 200rpm constant temperature oscillation shaking tables Bacterium solution can stop cultivating in cloud.3mL bacterium solution after culture is poured into the microcentrifugal tube of 1.5mL in two times, twice It is centrifuged 1min respectively with 12000rpm, is centrifuged all discards supernatant solution twice, retains precipitating.Add into the precipitating after being centrifuged twice The TSB culture medium for entering 50 μ L sufficiently suspends precipitating, adds the pMAL-C4x-PFc recombinant plasmid of 3 μ L and the KCM solution of 10 μ L It mixes well, in addition takes the 100 μ L system of non-resistant LB liquid medium polishing of 37 μ L.It is placed at room temperature for after ice bath 20min 10min, in the non-resistant LB liquid medium that 500 μ L are added in super-clean bench into above-mentioned microcentrifugal tube, in 37 DEG C of 200rpm 15min is cultivated on constant temperature oscillation shaking table, 5000rpm is centrifuged 5min later, it is resuspended with the non-resistant LB liquid medium of 200 μ L, The LB solid culture ware of 100 μ L bacterium solution tripods coating ammonia benzyl resistance is drawn, is inverted and is incubated overnight in 37 DEG C of constant temperature incubators, see Examine bacterium colony growing state, remaining 100 μ L bacterium solutions be put in 4 DEG C it is spare.

(5) determination of the inducing expression and condition of recombinant bacterium

It is dried on gum-making rack after the slab of the configuration PAGE gel of 1.0mm and thin plate are cleaned, uses agar powder The solution of configuration 2% carries out back cover, the SDS- with glue specification configuration 12% provided according to green skies biotechnology research institute PAGE gel be put in 4 DEG C it is spare.

A. relatively suitable expression temperature is explored

In the LB liquid medium of 5mL ammonia benzyl resistance, (ampicillin concentration is 60 μ g/ to white pipette tips picking positive BL21 bacterium colony ML in 37 DEG C of 200rpm constant temperature oscillation shaker overnight culture about 12h in), drawn from the bacterium solution being incubated overnight 300 μ L in 37 DEG C of 200rpm constant temperature oscillation shaking tables in the 2YT fluid nutrient medium (ampicillin concentration is 60 μ g/mL) of new 5mL ammonia benzyl resistance 2~3h is cultivated, when OD600 value being made probably to reach 0.6~0.8, the 50 μ L of IPTG solution of 100mmol/L is added thereto, makes The final concentration of 1.0mmol/L of IPTG, then at 25 DEG C, 30 DEG C and 37 DEG C, inducing expression 4h respectively, while with not converting The empty BL21 bacterium of pMAL-C4x-PFc recombinant plasmid is as a control group.Ammonia benzyl positive BL21 bacterium after taking above-mentioned inducing expression respectively 5 × SDS-PAGE sample-loading buffer of liquid and each 80 μ L of sky BL21 bacterium solution and 20 μ L are sufficiently mixed, 100 DEG C of heating water bath 10min After to be down to room temperature spare.Configured 12% SDS-PAGE glue is put into protein electrophorese instrument, the albumen newly configured is added Electrophoretic buffer carefully extracts comb out of glue, by the albumen Marker point of each sample 20 μ L and 5 μ L into sample Hole carries out SDS-PAGE electrophoresis and appropriate destainer is added in micro-wave oven after the equal level dyeing 1h of coomassie brilliant blue R_250 dyeing liquor Heating decoloration, changes a destainer every 5min, is analyzed with gel imaging system according to glue after successfully decolourizing.If its success table It reaches, then the relatively large number of inducing temperature of further screening expression quantity, while saving the recombination bacterium solution before inducing expression with glycerol 1mL。

As shown in Fig. 2, M is classified as albumen Marker in figure, A is classified as sky BL21 bacterium solution in figure, and B is classified as inducing temperature and is in figure 25 DEG C of ammonia benzyl positive BL21 bacterium solution, C is classified as the ammonia benzyl positive BL21 bacterium solution that inducing temperature is 30 DEG C in figure, and D, which is classified as, in figure lures Lead the ammonia benzyl positive BL21 bacterium solution that temperature is 37 DEG C；Ammonia benzyl positive BL21 bacterium is added appropriate dense eventually at 25 DEG C, 30 DEG C and 37 DEG C After the inducer IPTG that degree is 1.0mmol/L induces 4h, observed through SDS-PAGE electrophoresis result, under the conditions of several temperature, recombination Albumen can be expressed, and the relatively large number of condition of expression quantity is 37 DEG C.

B. relatively suitable inducer concentrations are explored

Take the recombination bacterium solution being stored in glycerol in 30 μ L above-mentioned steps a that the LB Liquid Culture of the ammonia benzyl resistance of 5mL is added 37 DEG C of 200rpm constant temperature oscillation shaking tables are incubated overnight about 12h in base (ampicillin concentration is 60 μ g/mL), from what is be incubated overnight 300 μ L are drawn in bacterium solution 37 in the 2YT fluid nutrient medium (ampicillin concentration is 60 μ g/mL) of the ammonia benzyl resistance of new 5mL When OD600 value being made probably to reach 0.6~0.8, IPTG solution is added in DEG C 200rpm constant temperature oscillation 2~3h of shaking table culture thereto Making IPTG final concentration is respectively 0.1mmol/L, 0.5mmol/L, 1.0mmol/L, 1.5mmol/L, in the expression that upper step filters out Measure at 37 DEG C of relatively large number of temperature, inducing expression 4h, at the same with without conversion pMAL-C4x-PFc recombinant plasmid empty BL21 Bacterium is as a control group.5 × SDS-PAGE sample-loading buffer of each 80 μ L of bacterium solution and 20 μ L after taking above-mentioned inducing expression respectively fills Divide mixing, it is spare to be down to room temperature after 100 DEG C of heating water bath 10min.Configured 12% SDS-PAGE glue is put into protein In electrophoresis apparatus, the protein electrophoresis buffer that newly configures is added, by the albumen Marker point of each sample 20 μ L and 5 μ L into sample well SDS-PAGE electrophoresis is carried out, is observed with gel imaging system as a result, further exploring expression quantity relatively after dyeing and decoloration More induced concentrations.

As shown in figure 3, M is classified as albumen Marker in figure, A is classified as sky BL21 bacterium solution in figure, and B is classified as induced concentration and is in figure The ammonia benzyl positive BL21 bacterium solution of 0.1mmol/L, C is classified as the ammonia benzyl positive BL21 bacterium solution that induced concentration is 0.5mmol/L, figure in figure Middle D is classified as the ammonia benzyl positive BL21 bacterium solution that induced concentration is 1.0mmol/L, and E is classified as the ammonia that induced concentration is 1.5mmol/L in figure Benzyl positive BL21 bacterium solution；For ammonia benzyl positive BL21 bacterium at 37 DEG C, it is respectively 0.1mmol/L, 0.5mmol/ that appropriate final concentration, which is added, L, it after the inducer IPTG induction 4h of 1.0mmol/L, 1.5mmol/L, is observed through SDS-PAGE electrophoresis result, several inducers are dense Under the conditions of degree, recombinant protein can be expressed, and the relatively large number of condition of expression quantity is that inducer IPTG concentration is 0.5mmol/L。

C. relatively suitable expression time is explored

Take the recombination bacterium solution being stored in glycerol in 30 μ L above-mentioned steps a that the LB Liquid Culture of the ammonia benzyl resistance of 5mL is added It is incubated overnight about 12h in 37 DEG C of 200rpm constant temperature oscillation shaking tables in base (ampicillin concentration be 60 μ g/mL), from being incubated overnight Bacterium solution in draw 300 μ L in the 2YT fluid nutrient medium (ampicillin concentration be 60 μ g/mL) of the ammonia benzyl resistance of new 5mL In 37 DEG C of 200rpm constant temperature oscillation 2~3h of shaking table culture, when OD600 value being made probably to reach 0.6~0.8, in the expression explored Measure at relatively large number of IPTG concentration 0.5mmol/L and 37 DEG C of inducing temperature, respectively inducing expression 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, while as a control group with the empty BL21 bacterium without conversion pMAL-C4x-PFc recombinant plasmid.Above-mentioned induction table is taken respectively 5 × SDS-PAGE sample-loading buffer of each 80 μ L of bacterium solution and 20 μ L after reaching is sufficiently mixed, and is dropped after 100 DEG C of heating water bath 10min It is spare to room temperature.Configured 12% SDS-PAGE glue is put into protein electrophorese instrument, the protein electrophoresis newly configured is added The albumen Marker point of each sample 20 μ L and 5 μ L are carried out SDS-PAGE electrophoresis into sample well, after dyeing and decoloration by buffer It is observed with gel imaging system as a result, further exploring the relatively large number of induction time of expression quantity.

As shown in figure 4, M is classified as albumen Marker in figure, A is classified as sky BL21 bacterium solution in figure, and B is classified as induction time and is in figure The ammonia benzyl positive BL21 bacterium solution of 1h, C is classified as the ammonia benzyl positive BL21 bacterium solution that induction time is 2h in figure, when D is classified as induction in figure Between be 3h ammonia benzyl positive BL21 bacterium solution, E is classified as the ammonia benzyl positive BL21 bacterium solution that induction time is 4h in figure, and F, which is classified as, in figure lures The ammonia benzyl positive BL21 bacterium solution that the time is 5h is led, G is classified as the ammonia benzyl positive BL21 bacterium solution that induction time is 6h in figure, H column in figure The ammonia benzyl positive BL21 bacterium solution for being 7h for induction time, I is classified as the ammonia benzyl positive BL21 bacterium solution that induction time is 8h in figure；Ammonia benzyl Positive BL21 bacterium at 37 DEG C, be added inducer IPTG difference inducing expression 1h, 2h of appropriate final concentration of 0.5mmol/L, 3h, It after 4h, 5h, 6h, 7h, 8h, is observed through SDS-PAGE electrophoresis result, under several inducer time conditions, recombinant protein can be obtained It must express, the relatively large number of condition of expression quantity is that inducer IPTG induction time is 5h.

(6) great expression of recombinant protein

Take the recombination bacterium solution being stored in glycerol in 30 μ L above-mentioned steps (5) a that the LB liquid training of the ammonia benzyl resistance of 5mL is added It supports 37 DEG C of 200rpm constant temperature oscillation shaking tables in base (ampicillin concentration is 60 μ g/mL) and is incubated overnight about 12h, after culture 2mL is drawn in bacterium solution 37 in the 2YT fluid nutrient medium (ampicillin concentration is 60 μ g/mL) of the ammonia benzyl resistance of new 500mL DEG C 200rpm constant temperature oscillation 2~3h of shaking table culture, when OD600 value being made probably to reach 0.6~0.8, in the table that step (5) is explored Up to measuring under relatively large number of 37 DEG C of condition, the final concentration of 0.5mmol/L of inducer IPTG, induction time 5h, inducing expression is recombinated Bacterium, 6000rpm discards supernatant after being centrifuged 10min, with the 0.05mol/L Tris-HCL protein purification combination equalizing and buffering of 5mL Precipitating is resuspended in liquid, is crushed thallus with Ultrasonic Cell Disruptor in ice bath, and the every ultrasound interval 5s 3s ensures to radiate sufficiently, reduction because Ultrasonication and the amount of protein degraded.Ultrasound is to the good translucency of bacteria liquid has, and bacterium solution 6000rpm is centrifuged after ultrasound 10min collects precipitating and supernatant, is resuspended and is precipitated with 5mL Tris-HCL protein purification combination equilibration buffer, is turned with no Change the empty BL21 bacterium of pMAL-C4x-PFc recombinant plasmid as a control group.It is heavy after the supernatant of bacterium solution, resuspension after collection ultrasound It forms sediment and the albumen Marker of sky BL21 bacterium each 20 μ L and 5 μ L carries out SDS-PAGE electrophoresis, verify whether that successful expression recombinates egg White and albumen solubility.

As shown in figure 5, M is classified as albumen Marker in figure, A is classified as sky BL21 bacterium, and B is classified as sky BL21 bacterium, after C is classified as ultrasound The supernatant of bacterium solution, D are classified as the precipitating after being resuspended；In 37 DEG C of the relatively large number of condition of expression quantity that step (5) is explored, induce Under the final concentration of 0.5mmol/L of agent IPTG, induction time 5h, inducing expression recombinant bacterium, ultrasonication thallus, it is heavy to be collected after centrifugation Shallow lake and supernatant are resuspended with protein purification combination equilibration buffer and are precipitated, and express shape through SDS-PAGE electrophoretic analysis recombinant protein Formula, recombinant protein obtains great expression as the result is shown, and the recombinant protein in supernatant is more than the protein content in precipitating re-suspension liquid.

(7) purifying of recombinant protein: Dextrin Beads 6FF affinity chromatography purification of recombinant proteins is selected, according to normal State people from world and biotech firm's specification, first by Dextrin Beads 6FF be packed into chromatographic column in, with combine equilibration buffer into Adequately balance, the deposit sample after the resuspension in the step of handling (6) will be filtered with 0.45 μm of filter membrane carry out loading to row Processing carries out wash-out recombinant protein after sufficiently washing with miscellaneous buffer is washed, collects efflux, washes miscellaneous liquid and eluent.Take ultrasound Afterwards the supernatant, efflux of bacterium solution, wash miscellaneous liquid, eluent and sky BL21 bacterium each 20 μ L and 5 μ L albumen Marker carry out Result is observed with gel imaging system after SDS-PAGE electrophoresis, dyeing and decoloration.The recombinant protein that successful purification need to be gone out is long-term It is spare to be stored in -80 DEG C of refrigerators.

As shown in fig. 6, M is classified as albumen Marker in figure, A is classified as sky BL21 bacterium, and B is classified as the supernatant of bacterium solution after ultrasound, C It is classified as efflux, D, which is classified as, washes miscellaneous liquid, and E is classified as eluent；Select Dextrin Beads 6FF affinity chromatography purifying recombination egg It is white, according to Changzhou world people and biotech firm's specification, after filling column, balance, loading, washing and elution, collect respectively efflux, Cleaning solution and eluent take each sample to carry out SDS-PAGE electrophoresis, wash in efflux there is also a large amount of recombinant proteins as the result is shown Ingredient is more single in de- liquid, the recombinant protein predominantly purified.

This patent is attempted to select prokaryotic expression giant panda IgG Fc constant region for the first time, although Escherichia coli protokaryon Expression system, which has, cultivates the advantages such as easy, quick, economical and practical, but utilizes the recombinant protein of the expression system inducing expression More than half exists in the form of inclusion body, and soluble protein is only 13.3%~23% that summary table reaches albumen.This patent is selected MBP fusion tag promotes the great amount of soluble expression of giant panda IgG Fc segment, and being obtained using chemical transformation can express The recombination bacillus coli of giant panda IgG Fc segment, while convenient for utilizing the isolated purer recombinant protein of affinity chromatography. The recombinant vector pMAL-C4x-PFc containing giant panda IgG Fc fragment gene built is converted into BL21 bacterium, by inducing item The optimization of part has determined that the relatively suitable condition of the expression of the recombinant bacterium containing giant panda IgG Fc segment is 37 DEG C, IPTG concentration 0.5mM, inducing expression 5h successfully realize the great amount of soluble expression of giant panda IgG Fc segment.Utilize Dextrin Beads 6FF affinity protein purification has obtained purer soluble recombinant protein, convenient for preparation giant panda specificity antiantibody in next step.

Two, the recombinant protein of the prokaryotic expression of giant panda IgG Fc segment prepares the method for polyclonal antibody and polyclonal The detection of antibody

1, material

(1) experimental animal and serum

SPFKM healthy male mice 8 for testing 3~4 week old of Company of Animals Ltd. up to rich fruit purchased from Chengdu；Giant panda blood Clearly；Healthy lowlenthal serum；Healthy chicken serum；Healthy Swine serum；Healthy rabbit anteserum.

(2) main agents

Freund's complete adjuvant and incomplete Freund's adjuvant are purchased from rain occasion glass station；10 × detergent, TMB and terminate liquid All purchased from MEDIAN company；Skimmed milk power is purchased from U.S. company BD；Horseradish peroxidase (HRP) marks mountain sheep anti mouse IgG ELIAS secondary antibody is purchased from ABclonal company.

(3) main solution

PBS buffer solution, 10% skimmed milk, antigen coat liquid.

(4) key instrument equipment

Micro oscillator, triple valve, constant incubator, ice machine, pure water meter, multiple tracks liquid-transfering gun, common liquid-transfering gun, high pressure Steam sterilization pan, centrifuge, refrigerator, microplate reader, electronic balance.

2, the method that the recombinant protein of the prokaryotic expression of giant panda IgG Fc segment prepares polyclonal antibody, including following step It is rapid:

(1) measurement of recombinant protein concentration

The protokaryon table of purer giant panda IgG Fc segment has been obtained using Dextrin Beads 6FF affinity protein purification The recombinant protein reached surveys its IOD value using protein gray analysis software I mage-Pro Plus, uses albumen Marker as ginseng According to, obtain the recombinant protein of purifying mean concentration be 265.85 μ g/mL.

(2) preparation of polyclonal antibody

SPFKM healthy male mice 8 of 3~4 week old are raised one week or so, prevents its stress reaction from causing to test It influences.Wherein 5 mouse are as test group, and 3 mouse are as a control group.To reach 100 μ g dosage according to first immunisation mouse Demand, take 2500 μ L purifying protein samples (the 0.4 μ g/ μ L containing recombinant protein) and 2500 μ L Freund's complete adjuvant mixing and emulsifyings, cream Change into instillation water 15s not scatter, test group mouse neck, chest, dorsal sc branch inject each about 150 μ L, so that test The recombinant protein immunizing dose of every mouse of group is 500 μ L/ (100 μ g containing recombinant protein), and each test group mouse injection is identical Dosage.Interval two weeks, the recombinant protein for taking out purifying from -80 DEG C of refrigerators carry out SDS-PAGE electrophoresis, utilize protein gray scale point Analysis software I mage-Pro Plus surveys its IOD value again, obtains the existing concentration of recombinant protein.Same procedure is incomplete with Freund Adjuvant prepares vaccine sample, phase Tongfang and dosage and mouse progress is immunized for 2 times, and every test mice injection volume is made to reach 100 μ g Left and right.It is spaced again two weeks, it, will recombinant protein and the progress of incomplete Freund's adjuvant mixed in equal amounts after purification in right amount using the above method 3 times immune.After 3 times 7 days immune, 8 eyeball of mouse blood samplings are placed at room temperature for 2~4h, to its natural coagulation, 1000rpm centrifugation 3min dispenses each mouse serum, and a part is placed in 4 DEG C for detecting in next step, and it is standby that another part is placed in -80 DEG C of long-term preservations With.

(3) detection of polyclonal antibody potency

The antigen coat liquid of 200 μ L is separately added into 12 hole of ELISA Plate first row, the anti-of 100 μ L is respectively added in remaining 84 holes Primordial covering liquid.It is separately added into the recombinant protein after purification of 4 μ L into 12 hole of first row, 100 μ L are drawn after mixing well and arrive this Next hole of column, each Leie time carry out doubling dilution, and each last hole of column discards 100 μ L after mixing, enzyme mark entire in this way ELISA Plate is encapsulated in 37 DEG C of insulating box culture 2h in hermetic bag, takes out and dry each hole by each hole of plate all containing the liquid of same volume Liquid draws PBS buffer solution in washing 3 times on micro oscillator with the volley of rifle fire, every time 3~5min.Each time after washing, it will wash Liquid removes completely as far as possible.10% skimmed milk that 200 μ L PBS are configured respectively is added to each hole of ELISA Plate with the volley of rifle fire, is put after sealing In cultivating 2h in 37 DEG C of constant incubators, same method dries after washing 3 times after taking-up.Add respectively to each hole of ELISA Plate first row Enter the PBS buffer solution of 200 μ L, remaining each hole is separately added into the PBS buffer solution of 100 μ L.The serum of 5 test group mouse respectively takes 8 μ L is adequately mixed, and 5 μ L are separately added into each hole of first row, are mixed well, and draws 100 μ L to next hole of each row, respectively Row successively carries out doubling dilution, last hole of each row discards 100 μ L after mixing, each Kong Jun of entire ELISA Plate is made to contain 100 μ L Mixed liquor.1h is cultivated in 37 DEG C of constant incubators after ELISA Plate sealing, washs 5 times with 1 × cleaning solution after taking-up, every time 3 ~5min.Mountain is marked to each hole addition diluted horseradish peroxidase of PBS buffer solution 1:5000 (HRP) after ELISA Plate drying 50 μ L of sheep anti-mouse igg ELIAS secondary antibody, in 37 DEG C of constant incubator culture 30min after sealing.It is equally washed with 1 × cleaning solution after taking-up Wash 5 times, every time 3~5min.The TMB of 50 μ L is added after drying to each hole, is bound 5~15min with masking foil, adds 50 μ L's Terminate liquid terminates reaction.Each hole OD450 value is read with microplate reader, repeats above-mentioned test 3 times, it is dilute to determine that recombinant protein is most preferably coated with Release concentration.

Recombinant protein doubling dilution after purification is coated with plank, carries out ELISA test, and microplate reader reads OD450 value, repeats Test 3 times, corresponding each hole OD450 value are averaged, and averagely OD450 value is up to 2.10 as the result is shown, appears in the hole D3, are calculated The best dilution ratio of recombinant protein is 1:400 after purification out, is shown in Table 2.

The measurement of the best peridium concentration of 2 recombinant protein of table

It is the purification of recombinant proteins after 1:400 that each hole of 96 orifice plates, which is added 100 μ L and is diluted to best peridium concentration dilution ratio, 10% skimmed milk, 37 DEG C of incubation 2h, after washing drying, to each hole of ELISA Plate first row are added in 37 DEG C of incubation 2h after washing It is separately added into the PBS buffer solution of 200 μ L, remaining each hole is separately added into the PBS buffer solution of 100 μ L.Each hole of first row sequentially adds 5 Test group and each 4 μ L of 3 control group mice serum, carry out doubling dilution respectively, last hole of each row discards 100 after mixing μ L makes each Kong Jun of entire ELISA Plate contain the mixed liquor of 100 μ L.37 DEG C of incubation 1h washing dryings are added after ELIAS secondary antibody is incubated for again Sufficiently washing drying, adds TMB to be protected from light colour developing, and terminate liquid is added and terminates reaction, and microplate reader reads OD450 value, repeats above-mentioned test 3 It is secondary, calculate the potency of the polyclonal antibody of preparation.

With the recombinant protein of the above-mentioned optimum diluting multiple dilution purifying explored, each hole of coated elisa plate, according to test Method carries out ELISA test, reads OD450 value with microplate reader, is repeated 3 times, and the identical each hole of dilution of each test group is averaged OD450 It is worth identical with control group each hole of dilution to be averaged the ratio between OD450 value, when its ratio is more than or equal to 2:1, maximum dilution multiple is For antibody titer, the polyclonal antibody potency for testing mouse No. 4 preparations is 1:51200, remaining 4 test mice prepares polyclonal Antibody titer is shown in Table 3 in 1:102400 or more.

The measurement (A450) of 3 polyclonal antibody potency of table

3, the detection of polyclonal antibody

The PBS buffer solution of 200 μ L is separately added into each hole of ELISA Plate first row, remaining each hole is separately added into the PBS of 100 μ L Buffer.Each hole of ELISA Plate first row is separately added into 4 μ L of giant panda serum, carries out doubling dilution, last hole of each row respectively 100 μ L are discarded after mixing, so that each Kong Jun of entire ELISA Plate is contained the mixed liquor of 100 μ L and are added after 37 DEG C of incubation 1h washing dryings 10% skimmed milk is coated with plank, is incubated for washing drying, each hole of first row is separately added into the test group mice serum 5 of mixed in equal amounts μ L, according to said determination, most preferably the operating method of recombinant protein peridium concentration carries out doubling dilution respectively after purification, uses microplate reader OD450 value is read, repeats above-mentioned test 3 times, determines giant panda serum and the coated optimum diluting multiple of mice serum.

It is coated with giant panda serum, carries out ELISA test, reads OD450 value with microplate reader, repeats test 3 times, corresponding each hole OD450 value is averaged, and averagely OD450 value is up to 2.13 as the result is shown, appears in the hole B2, calculates giant panda serum most Good dilution ratio is 1:100, and the best dilution ratio of mice serum is 1:80, is shown in Table 4.

4 giant panda serum of table is coated with elisa plate testing result

(1) detection of polyclonal antibody stability

For the stability of the polyclonal antibody of measurement preparation, carry out respectively batch in and batch between repeat to test.According to best dilute It releases ratio 1:80 and dilutes giant panda serum with antigen coat liquid, the serum of 8 mouse is diluted with PBS buffer solution, takes giant panda blood The two rows of each 100 μ L coated elisa plate of clear dilute sample are 24 holes, wash drying after normal incubation, 37 DEG C of 10% skimmed milk is added to incubate Drying is washed after educating 1h, each 100 μ L of every mice serum dilute sample is added separately in 3 holes, is washed and is got rid of after being sufficiently incubated for It is dry, add ELIAS secondary antibody to be incubated for, sufficiently plus enzyme substrate is protected from light colour developing after washing drying, adds terminate liquid and terminates reaction, microplate reader OD450 value is read, the interior stability of the polyclonal antibody batch of preparation is observed.

Acquisition test group mice serum is stored in 4 DEG C, repeats above-mentioned test daily, test 3 times is repeated altogether, with enzyme mark Instrument measures its OD450 value.Observe the stability with the extension of time, between polyclonal antibody batch.

In batch when test, the variance rate of every test group mouse 3 times balance measurement in primary test, maximum difference rate are calculated It is 4.00%, minimum difference rate is 2.19%.It repeats to test between batch, 3 test maximum difference rates of identical serum are 8.33%, most Small variance rate is 2.62%, is shown in Table 5.

The test result three times of 5 Stability Determination of table

(2) detection of polyclonal antibody specificity

It is diluted, healthy chicken, rabbit, pig and sheep blood serum according to upper according to the best dilution ratio 1:80 of giant panda serum The two rows of each coated elisa plate of detection operating method of polyclonal antibody stability are stated, it is normal to be incubated for washing drying, add 10% to take off Rouge cream is incubated for washing drying, and each 100 μ L of every mice serum dilute sample is added separately in 3 holes, washs after being sufficiently incubated for Drying, then plus ELIAS secondary antibody be incubated for, sufficiently washing drying after plus enzyme substrate develop the color, add terminate liquid terminate reaction, microplate reader Read OD450 value.Meanwhile giant panda serum carries out being coated with plank as a control test with after the dilution of equimultiple, microplate reader is read Take OD450 value.The OD450 value that chicken, rabbit, pig, sheep, giant panda each group are tested is observed, the specificity of polyclonal antibody is detected.

As shown in fig. 7, taking healthy chicken, rabbit, pig and sheep blood serum, ELISA is coated with according to giant panda serum optimum diluting multiple ELISA Plate carries out ELISA test, while being compared with giant panda serum, and test result is carried out with GraphPad Prism7 software Variance analysis between data, it is good with the polyclonal antibody and giant panda serum IgG reaction of mouse preparation as the result is shown, with There are cross reactivities for pig, sheep and rabbit serum, faint with chicken serum cross reaction.

This test is detected with polyclonal antibody potency, stability and specificity of the ELISA method to preparation.In order to Keep testing result as accurate and reliable as possible, this patent analyzes the various factors for influencing ELISA result, such as degreasing as much as possible The concentration of cream, the optimum response concentration of horseradish peroxidase-labeled mountain sheep anti-mouse igg ELIAS secondary antibody, the temperature of every step reaction and Time etc., and guarantee the ELIAS secondary antibody used, developing solution and terminate liquid etc. can normal reaction, reduce as far as possible non-specific Property combine etc. factors to test bring influence.Meanwhile in order to reduce non-specific binding, every step incubation terminates, repeatedly washs After drying, then carry out next step operation.

For potency, stability and the specificity of the polyclonal antibody that more accurately prepared by measurement, repeat 3 times Test, reduces the random error of test.Using batch in and batch between repeat the how anti-anti- with giant panda serum IgG of test discovery preparation Answering property is good, and test variance rate is lower, and polyclonal antibody stability is preferable.The antiantibody of preparation is deposited with pig, sheep and rabbit serum It is faint with chicken serum cross reaction in cross reactivity.This may be because with chicken comparatively, giant panda and pig, sheep and rabbit Son belongs to mammal, and affiliation is closer.

Affinity chromatography obtains the purer soluble recombinant protein immune health mouse that mean concentration is 265.85 μ g/mL, at Function is prepared for the giant panda polyclonal antibody that potency is high, stability is good.

Specific case used herein elaborates inventive concept, the explanation of above example is only intended to Help understands core of the invention thought.It should be pointed out that for those skilled in the art, not departing from this Under the premise of inventive concept, any obvious modification, equivalent replacement or the other improvements made should be included in the present invention Protection scope within.

Claims (10)

1. a kind of prokaryotic expression method of giant panda IgG Fc segment (PFc), which comprises the following steps:

(1) it constructs recombinant expression carrier: PFc being inserted between the BamH I and Hind III digestion site of pMAL-C4x carrier, Obtain pMAL-C4x-PFc recombinant vector；

(2) recombinant plasmid transformed bacillus coli DH 5 alpha bacterium, by pMAL-C4x-PFc recombinant vector obtained in step (1) and culture DH5 α bacterium be mixed, obtain expression PFc recombination DH5 α bacterium；

(3) extraction of recombinant plasmid: recombinating DH5 α bacterium obtained in ammonia benzyl resistance screening step (2), cultivates ammonia benzyl resistance screening Recombination DH5 α bacterium to OD600 value afterwards reaches 0.6-0.8, the recombination matter of the recombination DH5 α bacterium after culture is extracted using kit Grain, obtains pMAL-C4x-PFc recombinant plasmid；

(4) recombinant plasmid transformed glycerol stock BL21 bacterium: by pMAL-C4x-PFc recombinant plasmid obtained in step (3) and culture BL21 bacterium is mixed, and recombination BL21 bacterium is obtained；

(5) it recombinates the inducing expression of BL21 bacterium: recombinating BL21 bacterium obtained in ammonia benzyl resistance screening step (4) and cultivated Reach 0.6-0.8 to OD600 value, using the recombination BL21 bacterium after inducer inducing expression culture, protein purification is used after centrifugation It is resuspended and precipitates in conjunction with equilibration buffer, the recombination BL21 bacterium thallus after ultrasonication resuspension obtains table to bacteria liquid has translucency The recombinant protein reached.

2. the method for the prokaryotic expression of giant panda IgG Fc segment (PFc) according to claim 1, which is characterized in that step It suddenly further include the identification of recombinant plasmid in (3): the concentration and original pMAL-C4x-PFc weight of the recombinant plasmid of measurement recombination DH5 α bacterium The concentration of the recombinant plasmid of group carrier, using two kinds of restriction endonuclease cutting recombination DH5 α bacterium of BamH I and Hind III Recombinant plasmid and original pMAL-C4x-PFc recombinant vector recombinant plasmid, and measure cut after each recombinant plasmid plasmid it is dense Degree；And the recombinant plasmid and their BamH of the recombinant plasmid and original pMAL-C4x-PFc recombinant vector of DH5 α bacterium will be recombinated The digestion products of two kinds of restriction endonuclease of I and Hind III cutting carry out size comparison using nucleic acid electrophoresis.

3. the method for the prokaryotic expression of giant panda IgG Fc segment (PFc) according to claim 1, which is characterized in that step Suddenly in (5), using the recombination BL21 bacterium after inducer inducing expression culture, wherein inducing temperature is 25-40 DEG C.

4. the method for the prokaryotic expression of giant panda IgG Fc segment (PFc) according to claim 1, which is characterized in that step Suddenly in (5), using the recombination BL21 bacterium after inducer inducing expression culture, wherein inducer is IPTG, and inducer concentrations are 0.1-1.5mmol/L。

5. the method for the prokaryotic expression of giant panda IgG Fc segment (PFc) according to claim 1, which is characterized in that step Suddenly in (5), using the recombination BL21 bacterium after inducer inducing expression culture, wherein induction time is 1-8h.

6. the method for the prokaryotic expression of giant panda IgG Fc segment (PFc) according to claim 1, which is characterized in that step Suddenly in (5), ultrasonication be resuspended after recombination BL21 bacterium thallus when, ultrasound, and every ultrasound 4-6s are carried out in ice bath Interval 2-4s is abundant to ensure to radiate.

7. the method for the prokaryotic expression of giant panda IgG Fc segment (PFc) according to claim 1, which is characterized in that also Including step (6): the purifying of recombinant protein: using recombinant protein obtained in affinity chromatography purification step (5), wherein eluting It is the recombinant protein of purifying in liquid.

8. being obtained using the method for the prokaryotic expression of the described in any item giant panda IgG Fc segments (PFc) of claim 1-7 The method that recombinant protein prepares polyclonal antibody, which comprises the following steps:

S1: the measurement of recombinant protein concentration: the original of the giant panda IgG Fc segment of the isolated purifying of affinity chromatography is used The recombinant protein of nuclear expression is surveyed the IOD value of the recombinant protein of purifying using protein gray analysis software, uses albumen Marker As reference, the concentration value of the recombinant protein of purifying is obtained；

S2: the preparation of polyclonal antibody: by the recombinant protein of purifying and equivalent Freund's complete adjuvant mixing and emulsifying, emulsification to instillation 15s does not scatter in water, and the recombinant protein branch subcutaneous injection mouse after emulsification is immunized, every two weeks once, is carried out altogether It is immunized three times, wherein the injection volume of the preceding injection of mouse twice recombinant protein reaches 80-120 μ g, it is right 6-8 days after third time is immune Immune mouse carries out eyeball blood sampling, and blood is placed at room temperature for after its natural coagulation, carries out centrifuging and taking supernatant, that is, serum, obtains more grams Grand antibody.

9. the method according to claim 8 for preparing polyclonal antibody, which is characterized in that further include step S3: polyclonal The detection of antibody titer: mountain sheep anti-mouse igg ELIAS secondary antibody is marked using horseradish peroxidase (HRP), is inhaled using enzyme linked immunological Adhesion test (ELISA) determines that recombinant protein is most preferably coated with diluted concentration, the purifying after being diluted to best peridium concentration dilution ratio Recombinant protein carries out doubling dilution after mixing respectively with the serum of immune mouse, Non immune mouse after treatment, be incubated for washing Horseradish peroxidase (HRP) label mountain sheep anti-mouse igg ELIAS secondary antibody is added after drying to be incubated for, indirect enzyme-linked immunosorbent is used The potency of adsorption test (ELISA) calculating polyclonal antibody.

10. the giant panda polyclonal antibody that the method for preparing polyclonal antibody described in claim 8 or 9 prepares.