CN109735513B - Purification method of cryptosporidium protein kinase - Google Patents

Abstract

The invention discloses a purification method of cryptosporidium protein kinase, which comprises the steps of constructing an in vitro recombinant expression vector of the cryptosporidium protein kinase, performing plasmid transduction and IPTG low-temperature induced expression, and recovering purified protein through tapping after thallus cleavage after successful expression; the purification method of the invention improves the solubility of the expressed protein by means of low-temperature induction and improving the IPTG concentration, solves the problems of easy occurrence of self-cutting degradation inclusion body expression and the like in-vitro expression of cryptosporidium protein kinase by means of tapping recovery, can obtain recombinant protein with single high-purity strip and enzymatic activity, and can improve the purification effect on protein with difficult expression and larger molecular weight. The purified protein can be further used for functional analysis and research, and lays a foundation for solving a plurality of problems in the functional field of Cryptosporidium protein.

Description

A kind of purification method of cryptosporidium protein kinase

technical field

The invention relates to the field of biotechnology, and more specifically relates to a method for purifying an in vitro recombinant protein of Cryptosporidium protein kinase.

Background technique

Cryptosporidium ( Cryptosporidium ) is a widely distributed and serious cause of diarrheal diseases. Cryptosporidium is a parasitic protozoan of the Apicomplexan phylum, including more than 70 known species and genotypes. A disease that infects almost all vertebrates and causes diarrhea, especially persistent watery diarrhea in young animals. The infection rate of Cryptosporidium to young cattle, sheep and pigs is very high (>30%), and the cumulative infection rate to livestock within one year old is 100%. It is also the main pathogen of dairy cow diarrhea in many countries. Therefore, this disease causes serious harm to aquaculture.

In addition, Cryptosporidium is also an important zoonotic pathogen, and it is the second pathogen of children's diarrhea in developing countries. This pathogen accounts for 10% of children who die from diarrhea each year worldwide. Because of causing large-scale disease outbreaks, Cryptosporidium is the legal infectious disease pathogen in most developed countries, and it is also one of the only two pathogens in the water quality standards of many countries (including my country). The lack of effective drugs for cryptosporidiosis has resulted from the limited understanding of the invasion mechanism of Cryptosporidium. Among potential drugs for the treatment of cryptosporidiosis, only Nitazoxanide is the only drug approved by the US FDA, but this drug is ineffective in immunocompromised people and animals.

Protein kinases play important roles in the invasion and development of Apicomplexan protozoa, but their role in Cryptosporidium invasion has not been systematically studied. Apicomplexan has a conserved invasion mechanism. During this period, the secretion and activation of organelle proteins and the modification of host cell membranes and immune pathways by parasites are inseparable from the activation of protein kinases. Since the proteins of Cryptosporidium have not been purified, the functions of these proteins cannot be accurately verified.

For example, calcium-dependent protein kinase CDPK (Calcium-depending protein kinase) and insulin-degrading enzyme (insulin-degrading enzyme or IDE; insulin protease; insulysin; insulinase) are listed as potential drug targets. Studies have shown that the changes in the shape of the parasite, the rearrangement of the cytoskeleton, its recognition of the host cell, attachment, invasion, the discharge of some secreted proteins, and the escape of the parasite from the host cell are all closely related to the role of calcium ions. The processing and activation of these proteins and the completion of binding to host cell receptors require the synergy of multiple proteases, especially the participation of metalloproteases.

Because cryptosporidium protein kinases often appear in fragmented expression or self-cleavage degradation during the expression process in vitro, it is difficult to obtain relatively pure protein through conventional purification methods, and the activity cannot be guaranteed, which affects subsequent protein functions and enzymes. The scientific research has also hindered the systematic research on the invasion mechanism of Cryptosporidium.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the defects and deficiencies in the in vitro expression and purification process of the existing Cryptosporidium protein kinase, and provide a purification method of the Cryptosporidium protein kinase.

The first object of the present invention is to provide a method for purifying Cryptosporidium protein kinase.

Above-mentioned purpose of the present invention is given to realize by following technical scheme:

A method for purifying cryptosporidium protein kinase, comprising the steps of:

S1. Preparation of induced expression protein

S11. Constructing a prokaryotic expression vector containing the Cryptosporidium protein kinase gene;

S12. Transfer the prokaryotic expression vector of S11 into the expression host bacteria, pick the positive clones, and save them;

S13. The positive clones picked in S12 are expanded and cultured to the logarithmic growth phase, and then induced to express;

S14. Take the bacterial liquid after S13 induced expression, freeze and centrifuge to collect the bacterial cells, discard the supernatant, resuspend the bacterial cells, and then ultrasonically lyse the bacterial cells in an ice bath;

S15. Take the lysate obtained in S14, and centrifuge at low temperature to separate the supernatant and precipitate;

For soluble proteins, take the supernatant directly for subsequent experiments;

For insoluble proteins, wash and dissolve the precipitate with urea, refrigerate and centrifuge, and take the supernatant;

S16. Take the supernatant solution obtained in step S15 and filter it with a filter membrane of 0.45 μm, then incubate with the Ni column on ice, repeat the column several times, and then fully wash with the washing solution, then wash with 5 times the column volume. Deliquification to collect the target protein;

S2. Tapping rubber to recover purified protein

S21. Take the protein solution obtained in S16 for protein electrophoresis, and the process is carried out on ice; after electrophoresis, the protein gel is stained with ice-cold KCl solution for 5-10 minutes; after the development, cut off the milky white protein band at the size of the target protein according to the protein marker, Wash and soak in deionized water until colorless;

S22. Put the protein band obtained in S21 into a dialysis bag, add an appropriate amount of buffer solution (2 ml/1ug) to remove air bubbles, place the dialysis bag horizontally on a horizontal electrophoresis apparatus and soak it in the buffer solution, and perform electrophoresis at 4°C for 30~ 60 min, then change the electrodes, and continue electrophoresis for 2-3 min;

S23. Collect the protein solution obtained in the S22 dialysis bag and put it into a new dialysis bag, add buffer, and dialyze again to obtain purified Cryptosporidium protein kinase.

The inventors found that in the process of in vitro expression of Cryptosporidium protein kinase, fragmented expression or self-cleavage degradation occurred during the expression process. Conventional protein purification methods are difficult to obtain relatively pure and active proteins, which will affect subsequent protein production. Functional and enzymatic studies. The present invention solves the problems of self-cleavage, degradation and inclusion body expression in vitro expression of Cryptosporidium protein kinase through the rubber tapping recovery method, can obtain recombinant protein with single high-purity band and enzymatic activity, and can improve Purification of difficult-to-express, large-molecular-weight proteins. It should be noted that when the protein solution is subjected to protein electrophoresis in step S21, the sample must not be boiled at high temperature, and only needs to be treated with SDS loading buffer.

Preferably, the Cryptosporidium protein kinase includes but not limited to CpCDPK1 (Cryptosporidium calcium-dependent protein kinase 1) and CpCDPK9 (Cryptosporidium calcium-dependent protein kinase 9).

Specifically, the sequence of the CpCDPK1 gene is shown in SEQ ID NO: 1; the sequence of the CpCDPK9 gene is shown in SEQ ID NO: 6.

Preferably, the amplification primers of the CpCDPK1 gene are shown in SEQ ID NO: 1-2.

Preferably, the amplification primers of the CpCDPK9 gene are shown in SEQ ID NO: 4-5.

Preferably, the prokaryotic expression vector described in step S11 is pET-28a, and the expression host bacterium is Escherichia coli BL21.

Preferably, the inoculation amount of bacteria in step S13 is 1%, the OD600 value of the bacteria at the beginning of induction is between 0.6 and 0.8, and the conditions for inducing expression are IPTG with a final concentration of 0.2 to 0.4 mM (preferably 0.3 mM), 16 to 25 ℃, 150-200 rpm (preferably 180 rpm) for 4-6 h (preferably 5 h). The invention improves the solubility of the expressed protein by inducing low temperature and improving the concentration of IPTG.

Preferably, step S14 is specifically to collect the cells by centrifugation at 4°C, centrifuge at 8000 rpm for 15 min; resuspend the cells in PBS (add 10 ml PBS to every 200 ml of cell solution); The pause lasted 3 s for a total of 15 min. The process was carried out in an ice bath. The lysed bacterial solution was centrifuged at 4°C, 12000 rpm, for 30 min.

Preferably, in step S15, for the insoluble protein, the specific steps are as follows: take the precipitate and wash it with 2 M urea; add an appropriate amount of 8 M urea to the precipitate to dissolve (add 40 ml to 500 ml of bacterial solution), and first place it in an ice bath Incubate for 2 hours, then overnight at 4°C; centrifuge at 4°C, 12000 rpm, 30 min, and take the supernatant;

More preferably, in step S15, for insoluble proteins, if the urea solution does not hang on the column, a gradient urea elution method can be used instead of the subsequent column elution step, and the specific steps are as follows:

Resuspend the precipitate with 2 M urea solution, incubate on a shaker for 2 hours, 12000 rpm, 30 min, discard the supernatant, and collect the precipitate;

Continue to resuspend the collected precipitate with 4 M urea solution, incubate on a shaker for 2 hours, 12000 rpm, 30 min, discard the supernatant, and collect the precipitate;

Continue to resuspend the collected precipitate with 6 M urea solution, incubate on a shaker for 2 hours, 12000 rpm, 30 min, discard the supernatant, and collect the precipitate;

Finally, the pellet was resuspended with 8 M urea, incubated overnight on a shaker, 12000 rpm, 30 min, and the supernatant was collected for gel running verification.

Preferably, the Ni column purification in step S16 is specifically to wash the column with 5 times the column volume of deionized water and PBS respectively; add the Ni column to the filtered supernatant, and incubate on ice for 2-3 h; put the supernatant The solution was repeatedly applied to the column for 3 times, and all subsequent operations were carried out on ice; the eluent with PBS and low concentration of imidazole was used for sufficient elution, and the eluent with 5 times the column volume was used to collect the target protein.

Preferably, the concentration of the KCl solution in S21 is 0.2-0.3 M, and the temperature is 4°C.

Preferably, the conditions of the two forward and reverse electrophoresis in S22 are both at 4°C and a constant voltage of 100V.

Preferably, the volume ratio of protein solution to buffer in S23 is 1:15-25 (preferably 1:20).

Compared with the prior art, the present invention has the following beneficial effects:

The invention provides a method for purifying Cryptosporidium protein kinase. The purification method improves the solubility of the expressed protein through low-temperature induction and improving the concentration of IPTG, and at the same time solves the problem of in vitro expression of Cryptosporidium protein kinase through the method of tapping and recovering Problems such as self-cleavage, degradation and inclusion body expression that are prone to occur in the process can obtain recombinant proteins with a single band of high purity and enzymatic activity, and can improve the purification effect of proteins that are difficult to express and have large molecular weights. The purified protein can be further used for functional analysis research, laying the foundation for solving many problems in the field of Cryptosporidium protein function.

Description of drawings

Figure 1 is the result of electrophoresis of the CpCDPK1 gene fragment amplified in Example 1.

Fig. 2 is the verification result of pET28a-CpCDPK1-BL21 in Example 1.

Fig. 3 is the electrophoresis diagram of the protein after expression induced by the pET28a-CpCDPK1-BL21 plasmid in Example 1.

Fig. 4 is the electrophoresis diagram of the protein purified in Example 1.

Fig. 5 is the enzyme activity determination result of the protein purified in Example 1.

Fig. 6 is the result of electrophoresis of the CpCDPK9 gene fragment amplified in Example 2.

Fig. 7 is the verification result of pET28a-CpCDPK9-BL21 in Example 2.

Fig. 8 is the electrophoresis diagram of the protein after expression induced by pET28a-CpCDPK9-BL21 plasmid in Example 2.

Fig. 9 is the electrophoresis diagram of the protein purified in Example 2.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

Example 1

A method for purifying Cryptosporidium calcium-dependent protein kinase 1 (CDPK1) protein, comprising the following steps:

1. Construction of CpCDPK1 protein recombinant expression system

Using Cryptosporidium DNA as a template, specific amplification primers (SEQ ID NO: 1-2) were designed, and the CpCDPK1 gene fragment (SEQ ID NO: 3) was amplified by PCR. The electrophoresis of the amplified product is shown in Figure 1 .

The amplified CpCDPK1 gene fragment and the pET-28a plasmid were digested with EcoRI and XhoI, and the digested product was purified and ligated with T4 ligase to obtain the CpCDPK1 recombinant plasmid, which was then plated and single clones were picked. 1. Confirm the correctness of the recombinant plasmid after PCR sequencing verification, and name it pET28a-CpCDPK1.

2. Plasmid transduction and recombinant protein expression

The pET28a-CpCDPK1 plasmid was transferred into the expression strain BL21, cultured by streaking, and a single clone was picked. After PCR verification (Figure 2), the strain was preserved and named pET28a-CpCDPK1-BL21.

Transfer the expression strain successfully transferred to the recombinant plasmid into the medium for scale-up culture: configure 500 mL of LB medium, add kanamycin with a final concentration of 50 μg/mL after autoclaving and cooling, and pick pET28a -CpCDPK1-BL21 single colony was cultured overnight in shake flasks, and then inserted into 500 mL of fresh LB medium containing kana antibiotics at a 1% inoculum size to allow the bacteria to grow to the logarithmic growth phase.

When the bacteria grew to an _OD600 between 0.6 and 0.8, IPTG with a final concentration of 0.3 mM was added to induce expression. The induction conditions were 25°C, 200 rpm, and induced for 5 h; the electrophoresis results of the induced protein were shown in Figure 3 , indicating that the protein of interest is expressed at the expected size.

3. Purification of CpCDPK1 protein

Collect the pET28a-CpCDPK1-BL21 cells by centrifugation at 4°C, 8000 rpm, for 15 min, and discard the supernatant. Resuspend the cells with 25 mL of PBS, and lyse the cells by ultrasonic until the solution is clear and transparent. The ultrasonic working time is about 15 min, and the process is carried out in an ice bath. The lysate was centrifuged at 12,000 rpm for 30 min at 4°C, and the supernatant was collected and filtered through a 0.45 μm filter membrane.

Column elution, this process is carried out on ice:

Purification was performed using a 1 mL nickel column. Rinse the column with 5 times the column volume of deionized water and PBS respectively; then add the nickel column to the filtered supernatant and incubate on ice for 2-3 h; put the supernatant on the column repeatedly for 3 times. After the sample is completely combined with the Ni column, first wash the column with 5 times the column volume of PBS, then use 5 times the column volume of 20 mM imidazole solution, 40 mM imidazole solution, 60 mM imidazole solution to elute the impurity protein, and finally use 5 times the column volume Volume of 250 mM imidazole solution to collect the target protein, to obtain about 5 mL protein solution.

4. Recovery and purification of the target protein by rubber tapping

Add the collected protein solution into 5×SDS protein sample buffer at a ratio of 4:1, and high temperature boiling is prohibited during sample preparation. All samples were added to protein gel wells, and electrophoresis was carried out at a constant voltage of 120V for 60 min.

After electrophoresis, the protein gel was taken out, and the protein gel was stained with KCl solution with a concentration of 0.25 M pre-cooled to 4°C for 5-10 min. Tapping can be performed after the protein bands are clearly visible. According to the protein marker, intercept the milky white protein band at 70 kDa with a blade, put it into a beaker filled with deionized water at 4°C, wash and soak until colorless.

5. Electroelution

Put the protein band washed to colorless into a dialysis bag, add 10 mL PBS buffer, remove air bubbles, place it horizontally in a horizontal electrophoresis apparatus, and perform electrophoresis at 4°C and 100V constant voltage for 40 min. After the end, replace the electrodes and continue Perform electrophoresis for 2 min.

6. Dialysis

After electrophoresis, suck out 10 mL of the protein solution in the dialysis bag and put it into a new dialysis bag, put the dialysis bag into a beaker filled with 200 mL of PBS, dialyze in an ice bath for 1 hour, replace with fresh dialysate and then dialyze 1 h.

After dialysis, suck out 10 mL of the protein solution in the dialysis bag, and take 80 μL for gel running verification. The results of protein electrophoresis are shown in Figure 4. The first three lanes are protein samples without gel-tapping treatment, and the last two lanes are The purified protein samples obtained after the rubber tapping recovery process show that this process can well solve the problems of degradation and other problems in the protein purification process, and greatly improve the purity of protein samples. The remaining solution was divided into 1.5 mL centrifuge tubes and stored in a -80°C refrigerator.

7. Determination of enzyme activity

(1) Take the above-mentioned purified protein samples for enzyme activity detection, and the reaction system is shown in Table 1:

Table 1 CDPK1 protein sample enzyme activity detection reaction system

(2) Prepare 100 μL reaction solution and incubate at room temperature for 15 minutes.

(3) Add the purified protein sample to a final concentration of 10 nM, and mix quickly.

(4) Read on a microplate reader with a wavelength of 340 nm, a reading interval of 1 min, and a total time of 30 min.

The results of the enzyme activity assay are shown in Figure 5. The results show that this method can purify proteins such as protein kinases, and purified proteins with enzymatic activity can be obtained, and the protein samples are useful for further functional research.

In the above examples, the protein purification method provided by the present invention solves the problems of not single bands and impure proteins during the purification process.

Example 2

A method for purifying Cryptosporidium calcium-dependent protein kinase 9 (CDPK9) protein, comprising the following steps:

1. Construction of CpCDPK9 protein recombinant expression system

Using Cryptosporidium DNA as a template, the CpCDPK9 gene fragment (SEQ ID NO: 6) was amplified by PCR with specific primers (SEQ ID NO: 4-5). The electrophoresis of the amplified product is shown in FIG. 6 .

The amplified CpCDPK9 gene fragment and the pET-28a plasmid were digested with EcoRI and XhoI, and the digested product was purified and ligated with T4 ligase to obtain the CpCDPK9 recombinant plasmid, which was then plated and single clones were picked. 1. Confirm the correctness of the recombinant plasmid after PCR sequencing verification, and name it pET28a-CpCDPK9.

2. Plasmid transduction and recombinant protein expression

The pET28a-CpCDPK9 plasmid was transferred into the expression strain BL21, cultured by streaking, and a single clone was picked. After PCR verification (Figure 7), the strain was preserved and named pET28a-CpCDPK9-BL21.

Transfer the expression strain successfully transferred to the recombinant plasmid into the medium for scale-up culture: configure 500 mL of LB medium, add kanamycin with a final concentration of 50 μg/mL after autoclaving and cooling, and pick pET28a -CpCDPK9-BL21 single colony was cultured overnight in a shake flask, and then inserted into 500 mL of fresh LB medium containing kana antibiotics at a 1% inoculum size to grow the bacteria to the logarithmic growth phase.

When the bacteria grew to an OD ₆₀₀ between 0.6 and 0.8, IPTG with a final concentration of 0.5 mM was added to induce expression. The induction conditions were 16°C, 200 rpm, and induced for 5 h; the results of protein electrophoresis after induction were shown in Figure 8 The first two lanes are the empty plasmid control and the uninduced control respectively, and the last lane is the recombinant plasmid, indicating that the target protein is expressed at 130kDa.

3. Purification of CpCDPK9 protein

The pET28a-CpCDPK9-BL21 cells were collected by centrifugation at 4°C, 8000 rpm, for 15 min, and the supernatant was discarded. Resuspend the cells with 25 mL of PBS, and lyse the cells by ultrasonic until the solution is clear and transparent. The ultrasonic working time is about 15 min, and the process is carried out in an ice bath. The lysate was centrifuged at 4°C, 12000 rpm for 30 min, the supernatant was discarded, and the precipitate was collected.

The precipitate was dissolved with 40 mL of 8 M urea, and sonicated (sonication time 8 min), incubated in ice bath for 2 h, and incubated overnight at 4°C on a shaker. The lysate was centrifuged at 12000 rpm for 30 min at 4 °C, and the supernatant was collected and filtered through a 0.45 μm filter membrane. The filtrate was dialyzed against PBS through a dialysis bag before use.

Column elution, this process is carried out on ice:

Purification was performed using a 1 mL nickel column. Rinse the column with 5 times the column volume of deionized water and PBS respectively; then add the nickel column to the filtered supernatant and incubate on ice for 2-3 h; put the supernatant on the column repeatedly for 3 times. After the sample is completely combined with the Ni column, first wash the column with 5 times the column volume of PBS, then use 5 times the column volume of 20 mM imidazole solution, 40 mM imidazole solution, 60 mM imidazole solution to elute the impurity protein, and finally use 5 times the column volume Volume of 250 mM imidazole solution to collect the target protein, to obtain about 5 mL protein solution.

4. Recovery and purification of the target protein by rubber tapping

Add the collected protein solution into 5×SDS protein sample buffer at a ratio of 4:1, and high temperature boiling is prohibited during sample preparation. All samples were added to protein gel wells, and electrophoresis was carried out at a constant voltage of 120V for 60 min.

After electrophoresis, the protein gel was taken out, and the protein gel was stained with KCl solution with a concentration of 0.25 M pre-cooled to 4°C for 5-10 min. Tapping can be performed after the protein bands are clearly visible. According to the protein marker, intercept the milky white protein band at 70 kDa with a blade, put it into a beaker filled with deionized water at 4°C, wash and soak until colorless.

5. Electroelution

Put the protein band washed to colorless into a dialysis bag, add 10 mL PBS buffer, remove air bubbles, place it horizontally in a horizontal electrophoresis apparatus, and perform electrophoresis at 4°C and 100V constant voltage for 40 min. After the end, replace the electrodes and continue Run electrophoresis for 2 min

6. Dialysis

After electrophoresis, suck out 10 mL of the protein solution in the dialysis bag and put it into a new dialysis bag, put the dialysis bag into a beaker filled with 200 mL of PBS, dialyze in an ice bath for 1 hour, replace with fresh dialysate and then dialyze 1 h.

After dialysis, suck out 10 mL of the protein solution in the dialysis bag, and take 80 μL for gel running verification. The results of protein electrophoresis are shown in Figure 9. The first four lanes are protein samples that have not been treated with rubber tapping, and the last lane of the group is the protein samples that have been tapped. The purified protein samples obtained after the recovery process showed that this process can well solve the problems of fragmented expression during the protein purification process; the rest of the solution was divided into 1.5 mL centrifuge tubes and stored in a -80°C refrigerator.

sequence listing

<110> East China University of Science and Technology

South China Agricultural University

<120> A Purification Method of Cryptosporidium Protein Kinase

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gtggacgaag tcaccggcat tcataagtcc aactcaagcc ataattcaag tccaagttca 1980

aactctagct tagctcaaaa tcttcaccaa gaagaatcct tagcttcata tccaaagtac 2040

ttggatttca cagccccaga gttactcaaa ggagaaactt tctcctatag tcttgacgtg 2100

tggtctgttg gagttatcat gtattctgtc attackggac gccaccattt tagtggcagg 2160

tcacgagctg aaactaggca taatatctgc ttcggaattc ccgccatttc tgaaattaca 2220

tgcgtttcaa atgcatgcaa gtcatttatta ggaaagttac ttgagaaaaa tccaagagaa 2280

agaataacag tggatgaagc tttaagccat gattggtttt ctatttcgca ttccagttac 2340

tgcgaagttg atatagggat gcctctttta gcccacttga aagtttatac aagacagtca 2400

gacttgagac atatattaat tcatatgtta tcccatcaat tagcactaga tacaactcaa 2460

attaatatgg caaccagtgt ttttaaatct cttgataccg ataatgatgg agtattatca 2520

atttcagagt taggatctgg cttacaaaaa ttaggtattt catccagaga atcgtctatg 2580

atttattaaag ctatggatat tgatggtaat ggaattatat catatagtga atttatcaca 2640

gcttgtcata tttggagacg aaatgaaatt agacagttaa aatctttctt tatgaaaatt 2700

gatcgaaata atcagggaaa aattactaga gaagaattta aacagttgct taaggcacaa 2760

aaatctaaat tgctctcaaa tattaccaaa agcttttctt ctactaatga tgtcaattac 2820

agaacaattc ttccttcatc taatcaaggt gtatatactg aatgggattc tgttctagat 2880

gaaattgata ctaatagaga tggtcttatc gactggaaag aattctgtac ttttattgtc 2940

gattacttta atacttctag ggaatctttg agggcatgtt acaacaattc atctatatct 3000

aatatttctt ctactttttc tattgatcct ccatccgttg cagggctctc aaggagaaga 3060

gattctggta tttgccccgt tcaagaagct aaccagcacc ctccccctca tccaagccaa 3120

ctagattaa 3129

Claims (5)

1. A method for purifying cryptosporidium protein kinase, which is characterized by comprising the following steps:

s1, preparing inducible expression protein

S11, constructing a prokaryotic expression vector containing a cryptosporidium protein kinase gene;

s12, transferring the prokaryotic expression vector of the S11 into expression host bacteria, selecting positive cloning bacteria, and preserving;

s13, performing expansion culture on the positive clone bacteria selected in the S12 to the logarithmic growth phase, and performing induction expression;

s14, taking bacterial liquid after the induction expression of the S13, freezing and centrifuging to collect bacterial bodies, discarding supernatant, re-suspending the bacterial bodies, and then performing ice bath ultrasonic splitting on the bacterial bodies;

s15, taking the lysate obtained in the step S14, and freezing and centrifuging to separate supernatant and precipitate;

taking supernatant for direct subsequent experiments on soluble proteins;

washing and dissolving the precipitate with urea, centrifuging at low temperature, and collecting supernatant;

s16, filtering the supernatant solution obtained in the step S15 by using a filter membrane with the size of 0.45 mu m, incubating the filtered solution with a Ni column on ice, repeatedly loading the solution on the column for several times, fully washing the solution by using a washing solution, and collecting target proteins by using an eluent with the volume of 5 times of the column volume;

s2, rubber tapping recovery and purification of protein

S21, carrying out protein electrophoresis on the protein solution obtained in the step S16, wherein when the protein electrophoresis is carried out, a protein sample is not boiled; the process is performed on ice; after electrophoresis, the protein gel is dyed by KCl solution for 5 to 10 minutes; cutting off lactalbumin strips at the size of target protein according to a protein Marker after development is finished, and washing with deionized water to soak until the target protein is colorless; the concentration of the KCl solution is 0.25 and M, and the temperature is 4 ℃;

s22, placing the protein strips obtained in the S21 into a dialysis bag, soaking the dialysis bag into a buffer solution, carrying out electrophoresis for 30-60 min, exchanging electrodes, and continuing electrophoresis for 2-3 min; wherein the electrophoresis is two times of forward and reverse electrophoresis, and the conditions are 4 ℃ and the constant pressure is 100V;

s23, collecting the protein solution obtained in the S22 dialysis bag, putting the protein solution into a new dialysis bag, replacing buffer solution for infiltration, and dialyzing again to obtain purified cryptosporidium protein kinase;

the cryptosporidium protein kinase is CpCDPK1 or CpCDPK9, and the gene sequence of the CpCDPK1 is shown in SEQ ID NO:3, the CpCDPK9 gene sequence is shown as SEQ ID NO: shown at 6.

2. The purification method according to claim 1, wherein the prokaryotic expression vector of S11 is pET-28a, and the expression host bacterium of S12 is Escherichia coli BL21.

3. The purification method according to claim 1, wherein the inoculum size of the cells in the step of expanding culture in S13 is 1%, the OD600 value of the cells at the beginning of induction is 0.6-0.8, the conditions of induction expression are IPTG with a final concentration of 0.2-0.4 mM, 16-25 ℃, and induction is performed at 150-200 rpm of 4-6 h.

4. The purification method according to claim 1, wherein in step S15, for insoluble proteins, if urea solution does not hang up on the column, a gradient urea elution method can be used instead of the subsequent column-loading elution step, and the specific steps are as follows:

resuspension the precipitate with 2M urea solution, shaking table incubation for 2h, 12000 rpm,30 min, discarding supernatant, and collecting precipitate;

the collected precipitate was further resuspended in 4M urea solution, and after shaking incubation for 2h, 12000 rpm,

30min, discarding supernatant, and collecting precipitate;

the collected precipitate was further resuspended in 6M urea solution, and after shaking incubation for 2h, 12000 rpm,

30min, discarding supernatant, and collecting precipitate;

finally, the precipitate was resuspended with 8M urea, and incubated overnight with shaking table at 12000 rpm for 30min, and the supernatant was collected for running verification.

5. The method according to claim 1, wherein the volume ratio of the protein solution to the buffer in S23 is 1: 15-25.