CN111925433A - Outizua erythropolis IFN alpha protein clone expression and polyclonal antibody preparation - Google Patents

Abstract

The invention relates to the cloning expression and polyclonal antibody preparation of the red gull IFN alpha protein, belongs to the technical field of bioengineering, and provides the cloning expression and polyclonal antibody preparation of the red gull IFN alpha protein. When the rabbit is immunized, the Freund's complete adjuvant and the incomplete adjuvant are selected to be matched with protein for injection, and the polyclonal antibody can be prepared efficiently and quickly. The prepared antibody is verified by a jojoba test, the recombinant protein of the gene partial fragment of the Ozuku IFN alpha has better immunogenicity, and the antibody titer can reach 1: 8. can be better used for the subsequent research on the antivirus mechanism of the gull-red IFN alpha protein.

Description

Outizua erythropolis IFN alpha protein clone expression and polyclonal antibody preparation

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to the clone expression and polyclonal antibody preparation of the Ozuku IFN alpha protein.

Background

Migration is an instinctive activity of migratory birds, and migratory birds choose to migrate to different places to live in order to avoid the threat to the migratory birds caused by the environment and the climate. During migration, it is easily carried by parasites including bacterial and viral pathogens. Birds, as carriers of pathogens, pose potential threats to the survival of other easily-dangerous or endangered birds and even to the health of humans around habitats. Recently, it has been reported that outbreaks of avian influenza are closely related to migratory birds, and so far, avian influenza has been isolated from hundreds of wild birds. Besides avian influenza, migratory bird migration process also carries other pathogens such as avian influenza, infectious bronchitis and the like, and poses potential threats to poultry health around migratory sites.

The red-mouth gull is used as a waiting bird with frequent migration, a large number of the red-mouth gulls migrate from northern Siberian areas to southern China for overwintering every year in winter, people like to feed the red-mouth gull beside a lake, and the red-mouth gull is in close contact with the red-mouth gull. This easily increases the possibility of causing human gull co-morbidities, especially some serious human and animal co-morbidities which may be caused by red-mouth gulls in the course of migration, such as serious animal epidemic diseases like avian influenza. Although the large-scale epidemic situation of red-mouth gull is not yet exploded, reliable data of that red-mouth gull is infected with avian influenza virus in long-distance migration process are available. Therefore, the research on the antivirus mechanism of the autoimmune system of the red gull is very important, and the foundation is laid for preventing and controlling diseases in the future.

For the red-mouth gull, the related research of the immune system is not reported, the related research content is more, and the key point for selecting important research is more. Avian interferons are classified into class I, class II and class III. Among them, type I interferon appears earlier and has broad-spectrum antiviral action. Alpha interferon (IFN α) is a class I interferon, a highly active, multifunctional, bioactive glycoprotein produced by immune-related cells, has antiviral effects, and is not selective for inhibition of viral replication. The IFN alpha product has wide clinical application and is researched more in mammals and poultry, but the research on wild migrating birds such as the red gull IFN alpha is not reported yet.

Disclosure of Invention

In order to overcome the problems in the background art, the invention provides the clone expression and polyclonal antibody preparation of the red gull IFN alpha protein, partial fragments of the red gull alpha interferon gene are pseudocloned, a prokaryotic expression recombinant vector is constructed, the recombinant protein is obtained, and the polyclonal antibody prepared by the recombinant protein provides a theoretical basis for later antiviral activity and antiviral mechanism research of the red gull alpha interferon gene, so that a new path is opened for preventing and controlling the red gull epidemic disease, and the veterinary public health is deeply influenced.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a gull IFN alpha protein, the amino acid sequence of which is shown in SEQ ID NO. 2.

The invention provides a gene for coding a protein of red gull IFN alpha, wherein the gene codes an amino acid sequence shown as SEQ ID NO.2, or a nucleotide sequence of the gene is shown as SEQ ID NO. 1.

The invention provides a method for cloning and expressing Oncochilus erythropolis IFN alpha, which comprises the following steps:

1) separating red-mouth gull blood lymphocytes, extracting total RNA, and reversely transcribing the RNA into cDNA;

2) designing an amplification primer, and carrying out PCR amplification to obtain a partial fragment of the Ozuku IFN alpha gene;

3) connecting a pMD18-T vector with a purified IFN alpha gene partial fragment, transforming a connecting product to a competent cell DH5 alpha for culturing, extracting nucleic acid from a cultured bacterial solution, and performing double identification by adopting PCR (polymerase chain reaction) and EcoR I and Xho I double enzyme digestion methods to obtain a recombinant plasmid pMD18-T-IFN alpha-1;

4) the recombinant plasmid pMD 18T-IFN alpha-1 is cut by EcoR I and Xho I to obtain the target fragment IFN alpha-1.

Further preferably, the method for clonal expression of gull IFN α further comprises the following steps:

5) construction of recombinant prokaryotic expression vector

The target fragment of red gull IFN alpha-1 is inserted between EcoR I and Xho I enzyme cutting sites of pET32a (+) vector, and a recombinant prokaryotic expression vector pET32a-IFN alpha-1 is constructed.

Further preferably, in step 2), the amplification primer sequences are as follows:

F：GAA TTC CTG CTC CTC CTG ACG GCT；

r: CTC GAG CTA ATT GCA CAT GGT GCG GGT GAG, respectively; the amplified fragment has a size of 537 bp.

More preferably, in step 2), the PCR reaction conditions are: pre-denaturation at 95 ℃ for 2min and denaturation at 95 ℃ for 30 s; annealing at 60 deg.C for 30s, extending at 72 deg.C for 1min, and performing 35 cycles; 10min at 72 ℃.

The invention provides a preparation method of a red gull IFN alpha protein, which comprises the steps of transforming the prepared red gull IFN alpha prokaryotic expression vector into a Rosetta competent cell, and carrying out IPTG induced expression to obtain the red gull IFN alpha recombinant protein.

More preferably, the bacterial liquid OD₆₀₀When the value is about 0.5-0.6, the induced expression condition is that 1mmol/ml IPTG is added to induce expression for 5h at the temperature of 37 ℃.

The invention provides a preparation method of a gull red IFN alpha polyclonal antibody, which takes the obtained gull red IFN alpha protein as an antigen to immunize rabbits, and the inoculation method is as follows:

1) preparation of immunogen: mixing the protein with equal volume of Freund complete adjuvant during first-time immunization, performing ultrasonic emulsification on ice for about 5min, and performing ultrasonic emulsification on ice for about 30min after mixing the protein with equal volume of Freund incomplete adjuvant during second-time immunization and third-time immunization;

2) immunization procedure: firstly, the method avoids: complete adjuvant + antigen, the injection dosage of the antigen is 1mg per tube, and the injection is injected in plantar skin; and (2) avoiding: incomplete adjuvant + antigen, the injection dosage of the antigen is 1mg per mouse, and abdominal intradermal injection is carried out; and (3) three-step (I): incomplete adjuvant + antigen, the injection dosage of the antigen is 1.5 mg/mouse, and the injection is performed subcutaneously on the back and the abdomen;

3) 14 days after the three-immunization, carotid bleeding was performed, and the polyclonal antibody was obtained by separating the serum.

The invention provides a gull IFN alpha polyclonal antibody, which is prepared according to the method.

The invention has the beneficial effects that:

the invention provides the clone expression and polyclonal antibody preparation of the red gull IFN alpha protein, clones partial fragments of the red gull alpha interferon gene, constructs a prokaryotic expression recombinant vector, obtains the recombinant protein to prepare polyclonal antibody, provides a theoretical basis for later antiviral activity and antiviral mechanism research of the red gull alpha interferon gene, and develops a new path for preventing and controlling the red gull epidemic disease and has deeper influence on the public health of veterinarians.

In order to efficiently and quickly prepare the polyclonal antibody, when the rabbit is immunized, the Freund's complete adjuvant and the incomplete adjuvant are selected to be matched with protein for injection, so that the preparation rate of the antibody is improved. The prepared antibody is verified by a jojoba test, the recombinant protein of the gene partial fragment of the Ozuku IFN alpha has better immunogenicity, and the antibody titer can reach 1: 8. can be better used for the subsequent research on the antivirus mechanism of the gull-red IFN alpha protein.

Drawings

FIG. 1 shows the results of the prediction of the Ozuku IFN α signal peptide.

FIG. 2 is an electrophoresis image of RNA extracted from lymphocytes.

FIG. 3 shows the result of PCR amplification of a partial sequence of IFN α gene.

FIG. 4 shows the partial sequence of pMD18-T-IFN α transformed into DH5 α.

FIG. 5 is a restriction verified clone; wherein, MDL2000, 1 is recombinant plasmid, 2, 3 are enzyme cutting.

FIG. 6 is PCR verified clones; among them, MDL15000, lane 1 is a PCR product.

FIG. 7 is a diagram of the recovery of pET32a carrier gel; wherein MDL15000, 1 is pET32a vector.

FIG. 8 IFN alpha-1 gel recovery; wherein, MDL2000, 1 is the recovery of IFN alpha-1 glue.

FIG. 9 shows the restriction enzyme verification of pET32a-IFN α -1; wherein, the holes 1 and 3 of MDL2000 are plasmids, and the holes 2 and 4 are enzyme cutting results.

FIG. 10 shows the expression of pET32a-IFN α -1 in an expression host.

FIG. 11 is an optimization of induction temperature; wherein, M: marker, lanes 1, 3, 5, 7, no IPTG treatment, and lanes 2, 4, 6, 8, 50ul IPTG treatment at 28 deg.C, 30 deg.C, 37 deg.C, 40 deg.C.

Figure 12 is an optimization of IPTG final concentration; wherein, M: marker, lane 1, no IPTG treatment, 2, 3, 4, 5, 6 IPTG induction at final concentrations of 0.25mmol/ml, 0.5mmol/ml, 1mmol/ml, 1.5mmol/ml, 2 mmol/ml.

FIG. 13 is an optimized induction time; wherein, M: marker, 1, 2, 3, 4, 5 for 2, 3, 4, 5, 6h induction, 6 for control wells.

FIG. 14 is the results of protein solubility analysis; wherein, M is marker, 1 hole is used for supernatant electrophoresis, and 2 holes are used for inclusion body electrophoresis.

FIG. 15 is the result of washing purification; wherein, M: marker, 1 is the unwashed result and 2 is the washing result of the washing solution.

FIG. 16 is a quantitative standard curve by the Broad method.

FIG. 17 is the results of the detection of antibody titers by the agar diffusion assay; wherein, 1, the serum titer is 1: 4; 2. the serum titer was 1: 4.

FIG. 18 shows the result of Western blot identification.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, preferred embodiments of the present invention will be described in detail below to facilitate understanding of the skilled person.

Example 1: analysis of biological information of Ozuku IFN alpha

Analyzing the basic physicochemical properties of the protein by using a protein analysis tool Protparam; predicting the signal peptide of the amino acid sequence of the Ozuku IFN alpha by using SignalP4.1; the rare codons of the red gull interferon alpha gene were analyzed using the CBS website. The results were analyzed as follows:

(1) analysis using the protein analysis tool ProtParam alpha showed that the total number of amino acids in the protein was 192, and it was predicted that the molecular weight of the protein was about 21.93kD and the theoretical value of isoelectric point was 10.91. The atomic composition is C: 961; h, 1500; n is 312; 264 parts of O; 9, total number of atoms 3046, formula C961H1500N312O264S9, analysis of amino acid composition showed that the most abundant amino acids in the protein were Leu (14.1%), Ala (9.9%), and His (9.9%). And the least abundant amino acids Lys (0.5%) Glu (0.5%) and Tyr (0.5%). Wherein the number of hydrophobic amino acids (A, I, L, F, W, V, G, P, M) is 86 and accounts for about 44.79% of the total number of amino acids, the number of hydrophilic amino acids (N, C, Q, S, T, Y) is 61 and accounts for about 31.77% of the total number of amino acids, the number of acidic amino acids (D, E) is 8 and accounts for about 4.17% of the total number of amino acids, and the number of basic amino acids (K, R, H) is 37 and accounts for about 19.27% of the total number of amino acids.

(2) The results of signal peptide prediction of gull IFN α using the online program signalp4.1 show: the maximum values of C-, S-or Y-values in the sequence of the Oncorhynchus gull IFN α protein were all greater than the threshold values, and a signal peptide was present at amino acids 1-31 of the protein (see FIG. 1).

(3) Rare codon analysis of Ou alpha interferon

On-line analysis results showed that 21 rare codons including Arg 1 CGA, 1 CGG, 1 AGG, 1 AGA, Gly 1 GGA, 1 GGG, Pro 11 CCC, Thr 4 ACG were present in the open reading frame of the red gull α interferon gene, and contained 2 consecutive rare codons (CCCAGA, GGGCCC).

Example 2: cloning of partial fragment of Outiola rubescens IFN alpha and construction of expression vector

2.1 extraction of Total RNA from peripheral blood lymphocytes of Laribacter erythropolis

(1) Blood lymphocyte separation

Lymphocytes were isolated according to the instructions for use of the lymphocyte isolation tube, and the cell solution was adjusted to 5X10 using incomplete RPMI1640 (containing 10% calf serum culture medium)⁶One/ml, added to 6 well cell culture plates. 1600 ul/well, 3 gradients per sample (no peripheral wells are required for 6 wells or contamination is possible), 80ul concanavalin A at 5mg/ml per well was added and incubated in a 5% CO2 incubator at 37 ℃. Taking the growth phase after culturing for 24hCells were seeded in 96-well cell culture plates (100 ul/well cell suspension, i.e. cells need to be digested); the interferon inducer polylc was added at a final volume of 200ul per well.

(2) Extraction of red gull peripheral blood lymphocyte total RNA

Scraping cells (about 9ml) in a culture dish, collecting the cells in a centrifuge tube, and centrifuging the cells at 1500r/min for 10 min.

② transferring the centrifuged supernatant to a new enzyme-free centrifuge tube, storing the centrifuge tube in a refrigerator at 4 ℃ for later use, and leaving the precipitate.

③ sucking 900ul of supernatant to dissolve the precipitate. Transferring the dissolved liquid into a 5ml new centrifugal tube, adding 2.7ml trizol, shaking, mixing, placing on ice, and freezing in a-20 deg.C refrigerator for 15 min. 720ul of chloroform was added, the centrifuge tube was closed, mixed well and ice-cooled for 15 min.

Fourthly, after centrifugation for 15min, the supernatant is mixed with isopropanol with the same volume as that of Trizol, the mixture is centrifuged after ice bath for 19min, 540ul of 75% ethanol is added after the supernatant is removed, and the mixture is centrifuged for 15min after uniform mixing.

Fifthly, pouring off the ethanol, opening the centrifugal tube cover, drying for 5min beside the alcohol lamp, and adding 21ul of DEPC water to dissolve the precipitate.

RNA reverse transcription

The reverse transcription is carried out by utilizing TaKaRa D6210A kit for reverse transcription of Baozhi, and the method refers to the kit instruction.

2.2PCR amplification of a partial fragment of the Ozuku IFN alpha Gene

(1) Primer design

According to the complete sequence of the red gull alpha interferon obtained in a laboratory, signal peptide prediction is carried out to obtain that the maximum value of C value, S value or Y value in the sequence of the red gull IFN alpha protein is larger than a critical value, and the protein contains signals at the amino acids 1-31. Therefore, in order to obtain a high-efficiency expression recombinant vector, the signal peptide is removed when designing a Primer, and an amplification Primer of a partial fragment of the interferon-alpha gene is designed by using Primer Premer5.0 software, wherein the sequence of the Primer is as follows:

F：GAA TTC CTG CTC CTC CTG ACG GCT；

r: CTC GAG CTA ATT GCA CAT GGT GCG GGT GAG, the amplified fragment has a size of 537 bp.

(2) PCR amplification of IFN alpha partial fragment

The PCR reaction system is shown in Table 1.

TABLE 1 PCR reaction System for partial fragment of IFN alpha Gene

The PCR amplification conditions are shown in Table 2:

TABLE 2PCR reaction conditions for IFN alpha Gene short sequences

And after the PCR amplification is finished, detecting the PCR product through agarose gel electrophoresis, and analyzing the amplification result.

2.3 cloning and identification of partial fragment T of Outizus erythropolis IFN alpha Gene

(1) Preparation of DH5 alpha

DH 5. alpha. competent cells were prepared according to molecular cloning, A laboratory Manual, third edition.

(2) Recovery of PCR product of part fragment of Outizus red IFN alpha gene

The test method was performed according to the instructions of the DNA recovery kit.

(3) pMD18-T vector was ligated to a partial fragment of purified IFN alpha gene

After partial fragment glue of pMD18-T and IFN alpha gene is recovered, the two fragments are connected, and the system is shown in Table 3:

TABLE 3 connection System

Referring to the loading system of the table, reagents were added to 0.2m1 EP tubes and ligated for 6h at 16 ℃.

(4) Transformation of ligation products into competent cells DH5 alpha

Firstly, the temperature of the water bath kettle is adjusted to 42 ℃.

② melting DH5 alpha, ice-cooling for 10min, adding 5ul ligation product, ice-cooling for 30 min.

③ water bath at 42 ℃ for 90s and ice bath for 5 min.

And adding 800 mul LB culture medium, and shaking for 1h in water bath at 37 ℃.

Fifthly, taking 100 mul of the conversion solution and spreading the conversion solution on an LB (AMP) solid culture dish. Before this operation, 40. mu.l of 2% X-gal, 8. mu.l of 20% IPTG were added dropwise to the plate to facilitate blue-white screening.

Placing the plate into a constant temperature incubator at 37 ℃ overnight.

Ninthly, selecting a single colony to be inoculated in 5ml LB/AMP, and carrying out water bath shaking culture at 37 ℃ for 12-16 h.

(5) Identification of recombinant plasmids

And extracting nucleic acid from the cultured bacterial liquid, and performing double verification by adopting pcr and a double enzyme digestion method.

② the PCR reaction system and reaction condition is the same as the amplification of partial IFN alpha gene segment.

② the two enzyme digestion identification of EcoR I and Xho I, the enzyme digestion system is shown in Table 4, the reaction condition is 37 ℃, 2 h.

TABLE 4 reaction System for enzyme digestion

And thirdly, identifying the correct recombinant plasmid, sequencing and analyzing, and then naming as pMD18-T-IFN alpha-1.

The results were analyzed as follows:

1. extraction of total RNA from lymphocytes

Collecting peripheral blood of red gull, separating and culturing lymphocyte, extracting total RNA by trizol method with good extraction effect, performing agarose gel electrophoresis to obtain complete RNA structure diagram, as shown in FIG. 2.

2. PCR amplification of partial fragment of IFN alpha gene

And (3) carrying out PCR partial fragment amplification on the cDNA after reverse transcription, and after optimizing conditions, amplifying to obtain a sequence with the size basically consistent with that of the target fragment, wherein after sequencing analysis is carried out on the sequence, the number of sequence bases is 537 and is the same as that of the target fragment.

3. Cloning and identification of part fragment T of Outiola rubella IFN alpha gene

The IFN alpha partial fragment gene is connected to a pMD18-T vector, and after the gene is transformed into DH5 alpha competent cells, the cloned strain grows well, as shown in figure 4, white colonies are screened by blue white spots to be positive, and blue colonies are negative. After selecting bacteria and culturing, extracting plasmids according to the instruction of the kit, performing double verification by EcoR I, Xho I double enzyme digestion (figure 5) and PCR (figure 6), sequencing and identifying the plasmids to obtain a target gene, and naming pMD18-T-IFN alpha-1.

Example 3: construction of prokaryotic expression vector of part fragment of Outiola erythropolis IFN alpha gene and protein expression

3.1 construction of recombinant prokaryotic expression vector pET32a-IFN alpha-1

(1) Extraction and recovery of PET32a (+) and pMD 18T-IFN alpha-1 plasmids

Culturing the strains of PET32a (+) and pMD 18T-IFN alpha-1.

② the plasmid PET32a (+) and recombinant plasmid pMD 18T-IFN alpha-1 according to the following table 5 enzyme cutting system at 37 ℃ for 3 h.

TABLE 5 reaction System for enzyme digestion

Thirdly, glue recovery is carried out on the carrier pET32a (+) after enzyme digestion and the target fragment IFN alpha-1.

(2) Construction of recombinant expression strains

Firstly, pET32a (+) and the target fragment IFN alpha-1 are connected for 6h at 16 ℃ in the system shown in the table 6.

TABLE 6 connection System

② transformation

See 2.3(4) for transformation procedure.

Identification of recombinant expression plasmid

Recombinant plasmid DNA was extracted and subjected to PCR identification and double restriction enzyme (EcoR I and Xho I) identification under the same PCR conditions as in Table 4, and the restriction enzyme system was as shown in Table 7 below.

TABLE 7 reaction System for enzyme digestion

Fourthly, sequencing and analyzing the recombinant plasmid pET32a-IFN alpha-1 which is correctly identified.

4.2 expression of recombinant proteins

4.2.1 transformation of pET32a-IFN α -1 plasmid into expression competent cells

Extracting pET32a-IFN alpha-1 plasmid, taking 5ul, and respectively transforming to BL21DE3 and Rosetta.

4.2.2 culture and expression of recombinant expression strains

(1) The transformed single colonies were inoculated to LB/AMP (double antibody) and cultured overnight at 37 ℃.

(2) The following day, 300ul of the above culture solution was inoculated into 4.7ml LB/AMP (double antibody), and when the OD600 was about 0.5-0.6, IPTG was added for induction.

(3) Collecting the induced bacteria liquid, processing the sample, and detecting by SDS-PAGE gel electrophoresis.

4.2.3 optimization of recombinant protein expression conditions

(1) Selection of expression competence

The plasmid transformed into BL21DE3, Rosetta was induced to express as described above and the optimal expression competence was screened by SDS-PAGE.

(2) Optimization of induction temperature

After the optimal surface competence is determined, the induction culture is carried out for 5h at different induction temperatures (28 ℃,30 ℃,37 ℃ and 40 ℃) in the induction process, samples are respectively processed and then are detected by SDS-PAGE, and the optimal induction temperature is determined according to the expression quantity.

(3) Optimizing IPTG Final concentration

After the host bacteria and the temperature are optimized, IPTG (0.25mmol/L, 0.5mmol/L, 1.0mmol/L, 1.5mmol/L and 2.0mmol/L) with different final concentrations are respectively added in the induction process to be cultured for 5 hours at the optimal induction temperature, samples are respectively treated, SDS-PAGE electrophoresis detection is carried out, and the optimal concentration of the IPTG is determined according to the expression quantity.

(4) Optimization of Induction time

After the recombinant expression bacteria are subjected to recovery culture, 50ul IPTG is added, induction is carried out for 2 hours, 3 hours, 4 hours, 5 hours and 6 hours at the temperature of 37 ℃, and SDS-PAGE identification is carried out on the bacteria liquid in each time period respectively to determine the optimal induction time.

The results were analyzed as follows:

1. recovery of T32a vector and pMD18-T-IFN alpha-1 glue

After bacteria are recovered, plasmids are extracted by a small plasmid extraction kit. The plasmid is subjected to double enzyme digestion by EcoR I and Xho I and then gel recovery is carried out. The pET32a vector (FIG. 7) and IFN α -1 (FIG. 8) were recovered.

2. ET32a-IFN alpha-1 construction and identification

The pET32a vector and the red-mouth gull IFN alpha-1 are connected and transformed to DH5 alpha, a single colony is selected for culture, and after plasmid extraction, the vector construction is successful through the double enzyme digestion verification of EcoR I and Xho I, as shown in figure 9.

3. Expression of T32a-IFN alpha-1 in an expression host

The expression of pET32a-IFN alpha-1 identified as positive by enzyme digestion and sequencing in the host is shown in FIG. 10. 1 and 2 are the expression condition of empty pET32a vector on Rosetta, about 20kd of the expression target protein, 3 and 4 are the expression condition of pET32a-IFN alpha-1 transformed to BL21DE3, the size of the expression recombinant protein is about 40kd, and 5 and 6 are the expression condition of the expression protein transformed to Rosetta. The expression quantity of the recombinant vector on Rosetta is higher.

4. Optimization of expression conditions

From the above results, it was found that the recombinant vector has a high expression level on Rosetta, and therefore, it was selected as the best competence, and after culturing this strain, the optimal expression temperature was selected: after the bacteria are recovered and cultured, when the bacteria grow to the OD600 value of about 0.5-0.6, IPTG is added to induce for 5 hours at different inducing temperatures (28 ℃,30 ℃,37 ℃, 40 ℃), and the results are shown by 12% SDS-PAGE: the recombinant protein has higher expression level at 37 ℃, as shown in figure 11. IPTG final concentration optimization is carried out, and 1mmol/ml is the optimal expression final concentration according to the protein expression quantity, as shown in figure 12. When all the above conditions were selected, the induction time was optimized, and 2, 3, 4, 5, and 6 hours were selected as the induction time, and as a result, it was found that the recombinant protein was highly expressed at 5 hours of induction, as shown in fig. 13.

Example 4: purification of recombinant proteins

4.1 purification of the protein

4.1.1 protein solubility assay

Recovering the expression bacteria: the strain is dipped and cultured in 5ml of resistant liquid culture medium at 37 ℃ overnight, 4ml is added into 196ml of double-resistant culture medium, IPTG is added when OD600 is 0.6-0.8, the final concentration is 1mmol/ml, and induction is carried out for 5h at 37 ℃. Collecting bacterial liquid, centrifuging at 100000rpm for 10min, discarding supernatant, and collecting precipitate. The collected precipitate was suspended in 25ml of 50mmol/ml Tris-HCl (pH8.0), and the precipitate was sonicated on ice after adding lysozyme. After centrifugation, the supernatant was left, and after washing the precipitate, the precipitate was dissolved with 8M urea. The supernatant and the pellet solubilized after washing were examined by SDS-PAGE to investigate protein solubility.

4.1.2 protein renaturation

(1) Treatment of dialysis bags

Cutting the dialysis bag into 10cm, placing in a sodium bicarbonate EDTA solution with a certain concentration, boiling, and cleaning the dialysis bag with distilled water.

(2) When the dialysis bag is used, one end of the dialysis bag is tied by a rubber band or a thread rope, and a sample to be dialyzed can be loaded in the dialysis bag after the bag is determined to be leakproof. The beaker can be used during dialysis, and the beaker is filled with dialysate to ensure that the dialysis bag is submerged below the liquid level.

(3) The whole dialysis process is carried out in a refrigerator at 4 ℃, and the concentrations of the renaturation normal saline urea buffer solution are respectively as follows: 8mol/L, 6mol/L, 4mol/L and 2mol/L, and the interval time of liquid change is 2-3 h.

(4) And centrifuging the renatured protein solution, taking the supernatant, and detecting the renaturation condition by SDS-PAGE.

(5) Protein concentration determination

Protein concentration was determined according to Bradford quantification kit instructions.

The results were analyzed as follows:

1. solubility analysis during protein purification

The induction bacteria are subjected to Tris-HCl dissolution freeze thawing, then are subjected to ultrasonic crushing, are centrifuged to collect supernatant, are washed by washing liquid, are dissolved by 8M urea, and are analyzed by SDS-PAGE with the dissolved inclusion bodies, so that the target protein mainly exists in the inclusion bodies, and the result is shown in figure 14.

2. Protein washing purification results

Because the target protein exists in the inclusion body, urea gradient washing is needed during purification, 2M urea is firstly used for washing, 4M urea is used for washing, 8M urea is finally used for dissolving, and SDS-PAGE gel electrophoresis is carried out after the dissolved solution is treated by SDS sample loading liquid, so that a relatively single target protein band is obtained, as shown in FIG. 15.

3. Results of protein content determination by Broad method

The purified protein was stained with Coomassie brilliant blue staining solution, and then protein content was measured using NP80 nucleic acid protein analyzer, and first, a protein quantitative standard curve as shown in the following (FIG. 16) was obtained by reacting the standard sample with the staining solution. It can be seen from the figure that the regression equation of the protein quantitative curve is that y is 1.3144x +0.014, the linear regression coefficient is 0.9986, and the content of the protein is 1.3mg/ml after coomassie brilliant blue staining solution staining.

Example 5: preparation of polyclonal antibodies

4.3.1 protein-immunized rabbits

(1) Preparation of immunogen: mixing the protein with equal volume of Freund's complete adjuvant during first-time immunization, performing ultrasonic emulsification on ice for about 5min, and performing ultrasonic emulsification on ice for about 30min after mixing the protein with equal volume of Freund's incomplete adjuvant during second-time immunization and third-time immunization.

(2) Immunization procedure: healthy New Zealand young rabbits 3 were selected and immunized according to the procedure shown in Table 8. 14 days after the three-immunization, carotid bleeding was performed, and the polyclonal antibody was obtained by separating the serum.

TABLE 8 immunization procedure

4.3.2 agar-agar titer determination

After preparing 1% agar, pour into sterile petri dish, punch with gel puncher. Adding purified protein as antigen into the central hole, adding multiple times of diluted hyperimmune serum as antibody and negative serum as control into the peripheral holes, diffusing in a constant temperature incubator at 37 deg.C for 24h, and placing in a wet plate for diffusion.

4.3.3 crude purity of Rabbit anti-IgG

(1) Serum was centrifuged at 3000r/min for 30min to clarify it, transferred to a beaker, and placed on a magnetic stirrer to stir.

(2) A saturated Ammonium Sulfate (AS) solution (4.lmol/L) was prepared, and the serum was mixed with an equal amount of AS solution to precipitate the antibody.

(3) The precipitation process was repeated several times.

(4) After precipitation, the solution was centrifuged and the supernatant discarded. 150mmol/L PBS equivalent to 25% -50% of the stock solution was slowly added.

(5) The dissolved antibody was dialyzed against PBS solution at 4 ℃ in a refrigerator.

4.3.4 Wetern-blot for identifying protein biological activity

(1) Sample treatment: the purified protein was processed and ready for loading.

(2) Preparing glue: cleaning the rubber plate, drying in the air, uniformly mixing the prepared gel liquid, adding the gel liquid into the rubber plate, firstly preparing 12% separation gel, and then preparing 5% concentrated gel.

(3) Loading: and (3) installing an electrophoresis tank and gel, adjusting the concentration of the quantified protein sample to be equal, loading, arranging a protein Marker contrast in a left hole, and performing electrophoresis at 150V for 1 h.

(4) Film transfer: the PVDF membrane is cut into the size of gel, soaked in methanol for several minutes, and then transferred into electrotransformation liquid for soaking. 6 pieces of filter paper are cut out, the size of the filter paper is consistent with that of the gel, and the filter paper is also immersed in the transfer liquid. Unloading the glue, marking the upper left corner cut, and sequentially: placing the cathode, the gasket, the filter paper, the gel, the PVDF film, the filter paper and the gasket, placing the anode, adding the electric transfer liquid, closing the cover, placing the ice box, adjusting the constant voltage to 110V and 150mA, and taking out the ice box after 1.5 hours.

(5) And (3) sealing: the finished membranes were washed and blocked with 5% BSA for 2 h.

(6) In combination with an antibody: the membrane was washed 3 times with TBST and diluted primary antibody was added for binding at room temperature for 2 h.

(7) And (3) secondary antibody incubation: the membrane was rinsed 3 times with TBST and incubated with secondary antibody.

(8) And dyeing with DBA dyeing solution.

The results were analyzed as follows:

1. antibody titer results of agar-agar gel

The prepared hyperimmune rabbit antiserum is used as an antibody, the recombinant purified protein is used as an antigen, and the highest antibody titer can be obtained by agarose gel diffusion, wherein the highest antibody titer can be 1: 4. The results show that the recombinant protein can induce the rabbits to generate good immune response, as shown in FIG. 17.

2. Western blot results

The gull red IFN alpha protein is used as an antigen, a rabbit antiserum extracted roughly is used as a primary antibody, a goat anti-rabbit HRP labeled antibody is used as a secondary antibody, and western blot is adopted to identify the biological activity of the protein. The test result shows that the recombinant protein has better reactogenicity and can be specifically combined with the rabbit anti-positive serum, as shown by an arrow in figure 18.

The invention successfully constructs the IFN alpha gene partial fragment recombinant expression vector by taking pET32a (+) as a prokaryotic expression vector. In the research, Rosetta is suitable for expression under the condition of moderate temperature, and several temperatures of 28 ℃,30 ℃,37 ℃ and 40 ℃ are selected as expression screening conditions when the temperature is optimized, so that the result shows that the recombinant protein has the highest expression level under the condition of 37 ℃. In the experiment, IPTG sugar is used as an inducer, so that the large-scale expression of the downstream gene of the Lac promoter can be regulated, but the metabolic function of host bacteria is easily hindered by using IPTG excessively. Therefore, the optimum concentration of IPTG can be determined to avoid the influence of IPTG on thalli and ensure the expression amount of protein. The optimum induction concentrations were selected under the conditions of inducing protein expression of 0.25mmol/ml, 0.5mmol/ml, 1mmol/ml, 1.5mmol/ml and 2mmol/ml, and as a result, it was found that the protein expression level was the highest at a final concentration of 1mmol/ml, and thus the optimum expression concentration was selected. Time is also an important factor in the protein expression process, if the induction time is not enough, the protein can not be fully expressed, and on the contrary, if the induction time is too long, the expressed protein carries metabolites in the bacterial growth process, so that the purification process has certain difficulty. And (3) optimizing the protein expression time by taking 2, 3, 4, 5 and 6h, and finally determining that the protein expression amount reaches a peak value in 5h, so that the optimal induction time is determined to be 5 h. In order to prepare polyclonal antibody efficiently and quickly, when a rabbit is immunized, the Freund's complete adjuvant and incomplete adjuvant are selected to be matched with protein for injection, and the preparation rate of the antibody is improved. The prepared antibody is verified by a jojoba test, the recombinant protein of the gene partial fragment of the Ozuku IFN alpha has better immunogenicity, and the antibody titer can reach 1: 8. can be better used for the subsequent research on the antivirus mechanism of the gull-red IFN alpha protein.

Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.

SEQUENCE LISTING

<110> Yunnan university of agriculture

<120> red mouth gull IFN alpha protein clone expression and polyclonal antibody preparation

<130> 2020-07-28

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 579

<212> DNA

<213> Larus ridibundus

<400> 1

atgcctgcgc ccgcaaccca ccacacccgc ctgcgccacg gcgccccgac gctcctgctc 60

ctcctgacgg ctctcgccac caccctcgcc tgccgccacc tgcctccctg ccactccacc 120

ttcccctggg acagcctcag catcctccgg gacatggctc ccagaccgcc acagccctgc 180

caacaccaac aggcgccctt ccccttcccc gacaccctcc tccacaacag ccacccacag 240

caagccgccg ccaccgcccg acacatcctc gacaacctct tcgccaccct cagcgcacag 300

agcacccccc aacactggga cgaccaggca cgccatcgcc tcctcaacaa cctccaccgc 360

cacagccagc agctccagca atgccccgca cccaacggca cgctctccca aggacaaggg 420

ccccgcaacc gcacgctcac catcagcaaa tacttcaggc gcatccacaa cttcctccac 480

acccacaacc acagcgcctg cgcctgggac cacgtccgcc tcgaagttcg cgcctctttc 540

cagcgcctcc acaacctcac ccgcaccatg tgcaattag 579

<210> 2

<211> 192

<212> PRT

<213> Larus ridibundus

<400> 2

Met Pro Ala Pro Ala Thr His His Thr Arg Leu Arg His Gly Ala Pro

1 5 10 15

Thr Leu Leu Leu Leu Leu Thr Ala Leu Ala Thr Thr Leu Ala Cys Arg

20 25 30

His Leu Pro Pro Cys His Ser Thr Phe Pro Trp Asp Ser Leu Ser Ile

35 40 45

Leu Arg Asp Met Ala Pro Arg Pro Pro Gln Pro Cys Gln His Gln Gln

50 55 60

Ala Pro Phe Pro Phe Pro Asp Thr Leu Leu His Asn Ser His Pro Gln

65 70 75 80

Gln Ala Ala Ala Thr Ala Arg His Ile Leu Asp Asn Leu Phe Ala Thr

85 90 95

Leu Ser Ala Gln Ser Thr Pro Gln His Trp Asp Asp Gln Ala Arg His

100 105 110

Arg Leu Leu Asn Asn Leu His Arg His Ser Gln Gln Leu Gln Gln Cys

115 120 125

Pro Ala Pro Asn Gly Thr Leu Ser Gln Gly Gln Gly Pro Arg Asn Arg

130 135 140

Thr Leu Thr Ile Ser Lys Tyr Phe Arg Arg Ile His Asn Phe Leu His

145 150 155 160

Thr His Asn His Ser Ala Cys Ala Trp Asp His Val Arg Leu Glu Val

165 170 175

Arg Ala Ser Phe Gln Arg Leu His Asn Leu Thr Arg Thr Met Cys Asn

180 185 190

Claims (10)

1. A Ozuku IFN alpha protein, characterized in that: the amino acid sequence is shown in SEQ ID NO. 2.

2. A gene encoding a Outia erythropolis IFN alpha protein, characterized in that: the gene codes the amino acid sequence of the protein as claimed in claim 1, or the nucleotide sequence of the gene is shown as SEQ ID NO. 1.

3. A method for cloning and expressing Ovus pinicola IFN alpha is characterized in that: the method comprises the following steps:

1) separating red-mouth gull blood lymphocytes, extracting total RNA, and reversely transcribing the RNA into cDNA;

2) designing an amplification primer, and carrying out PCR amplification to obtain a partial fragment of the Ozuku IFN alpha gene;

3) connecting a pMD18-T vector with a purified IFN alpha gene partial fragment, transforming a connecting product to a competent cell DH5 alpha for culturing, extracting nucleic acid from a cultured bacterial solution, and performing double identification by adopting PCR (polymerase chain reaction) and EcoR I and Xho I double enzyme digestion methods to obtain a recombinant plasmid pMD18-T-IFN alpha-1;

4) the recombinant plasmid pMD 18T-IFN alpha-1 is cut by EcoR I and Xho I to obtain the target fragment IFN alpha-1.

4. The method of claim 3, wherein the expression of the gull IFN α clone is as follows: further comprising the steps of:

5) construction of recombinant prokaryotic expression vector

The target fragment of red gull IFN alpha-1 is inserted between EcoR I and Xho I enzyme cutting sites of pET32a (+) vector, and a recombinant prokaryotic expression vector pET32a-IFN alpha-1 is constructed.

5. The method for clonal expression of gull IFN α according to claim 3 or 4, wherein: in step 2), the amplification primer sequences are as follows:

F：GAA TTC CTG CTC CTC CTG ACG GCT ；

r: CTC GAG CTA ATT GCA CAT GGT GCG GGT GAG, respectively; the amplified fragment has a size of 537 bp.

6. The method for clonal expression of gull IFN α according to any of claims 3 to 5, wherein: in the step 2), the PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 1min, and 35 cycles; stretching at 72 deg.C for 10 min.

7. A preparation method of a red gull IFN alpha protein is characterized by comprising the following steps: transforming the prokaryotic expression vector of the gull IFN alpha prepared in the claim 4, 5 or 6 into Rosetta competent cells, and performing IPTG induced expression to obtain the recombinant protein of the gull IFN alpha.

8. The method of claim 7, wherein the method comprises: bacterial liquid OD₆₀₀When the value is about 0.5-0.6, the induced expression condition is that 1mmol/ml IPTG is added to induce expression for 5h at the temperature of 37 ℃.

9. A preparation method of a polyclonal antibody of Ovumgallus erythropolis IFN alpha is characterized by comprising the following steps: a rabbit immunized with the protein of Laribacter erythrorhizoides IFN α obtained according to any one of claims 1 to 8 as an antigen by the following method:

1) preparation of immunogen: mixing the protein with equal volume of Freund complete adjuvant during first-time immunization, performing ultrasonic emulsification on ice for 5min, mixing the protein with equal volume of Freund incomplete adjuvant during second-time immunization and third-time immunization, and performing ultrasonic emulsification on ice for 30 min;

2) immunization procedure: firstly, the method avoids: complete adjuvant + antigen, the injection dosage of the antigen is 1mg per tube, and the injection is injected in plantar skin; and (2) avoiding: incomplete adjuvant + antigen, the injection dosage of the antigen is 1mg per mouse, and abdominal intradermal injection is carried out; and (3) three-step (I): incomplete adjuvant + antigen, the injection dosage of the antigen is 1.5 mg/mouse, and the injection is performed subcutaneously on the back and the abdomen;

3) 14 days after the three-immunization, carotid bleeding was performed, and the polyclonal antibody was obtained by separating the serum.

10. A polyclonal antibody of Ozuku IFN alpha, which is characterized in that: the polyclonal antibody is prepared according to the method of claim 9.

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