CN106337054A - Method for preparing high activity recombinant porcine epidermal growth factor - Google Patents

Abstract

The invention discloses a preparation method of highly active recombinant porcine epidermal growth factor. The present invention firstly discloses the nucleotide sequence shown in SEQ ID NO.2 and its recombinant vector and recombinant bacteria. The method of the invention can efficiently express the recombinant porcine epidermal growth factor with a high rate, and the prepared recombinant porcine epidermal growth factor has high activity and has good industrial application prospect.

Description

A kind of preparation method of highly active recombinant porcine epidermal growth factor

technical field

The invention relates to the field of genetic engineering, in particular to a method for preparing a highly active recombinant pig growth factor.

Background technique

Epidermal growth factor (EGF) is a multifunctional growth factor, which has a strong mitogenic effect on various tissue cells both in vivo and in vitro. Epidermal growth factor binds to the receptor epidermal growth factor receptor (EGFR) on the cell surface and stimulates the activity of the receptor's intrinsic tyrosine kinase, thereby initiating a signal transduction cascade that leads to a variety of biochemical changes. Intracellular calcium Elevated levels, increased glycolysis and protein synthesis, increased expression of certain genes (including epidermal growth factor receptor), ultimately leading to DNA synthesis and cell proliferation. Epidermal growth factor is abundant in milk, and its receptors are widely distributed in the digestive tract, so it plays an important role in the development of the gastrointestinal tract of newborn animals.

In modern intensive breeding, the application of porcine epidermal growth factor can enhance the health level of young animals and enhance the overall production performance of animals. It is one of the means to fundamentally solve the contradiction between the rapid growth of pigs and the lag of organ development. Therefore, it is particularly important to artificially produce and prepare epidermal growth factor. Jiao Xiaojun, "Expression of porcine epidermal growth factor in high-density fermentation of recombinant Pichia pastoris and its purification and identification", Journal of South China Agricultural University, Volume 32, No. 1, January 2011, and Wang Yang et al. "Porcine epidermal growth factor in Pichia pastoris Expression and Identification", Chinese Agricultural Sciences 2007,40 (11): 2593-2599 all disclosed the method of using Pichia pastoris recombinant expression of porcine epidermal growth factor, but the activity of the recombinant porcine epidermal growth factor expressed was not high enough.

Contents of the invention

The purpose of the present invention is to provide a preparation method of highly active recombinant porcine epidermal growth factor.

The present invention provides the nucleotide sequence shown in SEQ ID NO.2.

The present invention also provides a recombinant vector, which includes the nucleotide sequence shown in SEQ ID NO.2.

Preferably, the recombinant vector is a recombinant Pichia pastoris expression vector pPIC9K.

The present invention also provides a recombinant bacterium, which is characterized in that it includes the aforementioned recombinant vector.

Preferably, the recombinant bacteria is recombinant Pichia pastoris.

The present invention also provides a fermentation method, which is characterized in that the aforementioned recombinant bacteria are cultured in a yeast culture medium and induced to express with methanol.

Preferably, it includes the steps of:

(1) Cell growth: Inoculate the recombinant bacteria into BMGY medium, culture at 25-35°C for 36-60 hours, and collect the cells by centrifugation;

(2) Induced expression: Then use BMMY induction medium to suspend the bacteria, and induce culture at 25-35°C for 48-120h.

Further preferably, in step (1), the culture temperature is 30°C, and the culture time is 48h; in step (2), the culture temperature is 30°C, and the induction time is 72h.

The present invention also provides the expression product prepared by the aforementioned method, such as the culture solution obtained by fermentation.

The present invention also provides a method for purifying recombinant porcine epidermal growth factor, the steps are as follows:

a. Take the aforementioned expression product, centrifuge and dialyze;

b. Chromatographic separation.

The chromatographic separation method is: first use Ultrasphere-ODS chromatographic column to separate, and then use C18 chromatographic column to separate.

The present invention also provides the purified product prepared by the aforementioned method.

The present invention also provides the use of the aforementioned expression product and the aforementioned purified product in the preparation of drugs or feed additives for improving the growth performance of piglets.

The method of the present invention obtains recombinant porcine epidermal growth factor, which has a very high activity, up to 3.97 times higher than that of commercially available hEGF, and the expression level is also higher. The eluate has high EGF activity and high content, is safe and has high nutritional value, and can be used to prepare drugs or feed additives for improving growth performance of piglets.

Apparently, according to the above content of the present invention, according to common technical knowledge and conventional means in this field, without departing from the above basic technical idea of the present invention, other various forms of modification, replacement or change can also be made.

The above-mentioned content of the present invention will be further described in detail below through specific implementation in the form of examples. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Description of drawings

The PCR result figure of Fig. 1 transformant, wherein, swimming lane 1,3,5,7,8,9,10,11,13,14,15,16,17,19,21,22 belong to positive clone, swimming lane 2, 4, 6, 12, 18, 20, and 23 belong to negative clones, M is a marker, and CK is a control.

The SDS-PAGE analysis of Fig. 2 expression product epidermal growth factor;

Figure 3 ELISA standard curve;

Fig. 4 EGF elution diagram;

Fig. 5 is a comparison diagram between the method of the present invention and the direct expression of porcine epidermal growth factor gene. M: Marker; Lanes 1, 3, 4, 7, 9, 11 are the expression of EGF gene, and lanes 2, 4, 6, 8, 10, 12 are the expression of EGF-n gene;

The chromatogram of Fig. 6 molecular sieve purification, add maker in the chromatogram, to mark the molecular weight of the product collected in the present invention;

Fig. 7 Effect of EGF on the growth rate of fibroblasts;

Fig. 8 Micrographs of cell growth at some concentrations.

detailed description

Example 1 Preparation of engineering bacteria containing target gene fragments

1. Acquisition of recombinant porcine epidermal growth factor gene

1. Gene sequence analysis

The known porcine epidermal growth factor gene was retrieved from the NCBI database, and the signal peptide part of the epidermal growth factor was removed at the same time to obtain the following sequence:

AATAGTTACTCTGAATGCCCGCCGTCCCACGACGGGTACTGCCTCCACGGTGGTGTGTGTATGTATATTGAAGCCGTCGACAGCTATGCCTGCAACTGTGTTTTTGGCTACGTTGGCGAGCGATGTCAGCACAGAGACTTGAAATGGTGGGAGCTGCGCTAA

The sequence is 162bp in total, encoding a protein with 53 amino acid residues:

nsysecppshdgyclhggvcmyieavdsyacncvfgyvgercqhrdlkwwelr

DNA Sequence Synthesis of Porcine Epidermal Growth Factor

The porcine epidermal growth factor gene sequence was analyzed, and a new EGF gene was designed and synthesized, named EGF-n (SEQ ID NO.2):

AATTCTTACTCTGAATGTCCACCTCTCCCACGACGGTTACTGTTTGCACGGTGGTGTTTGTATGTATATTGAAGCTGTTGATTCTTACGCTTGTAACTGTGTTTTTGGTTACGTTGGTGAAAGATGTCAGCATAGAGATTTGAAGTGGTGGGAACTGAGATAA

2. Construction of recombinant bacteria

1. Construction of pPIC-EGF-n vector

The whole gene EGF-n was cloned into Pichia pastoris constitutive expression vector pPIC9K through the EcoRI/NotI site to obtain the recombinant vector pPIC-EGF-n. The specific operation was carried out according to the recombination operation part of "Molecular Cloning Experiment Manual".

2. Electroporation transformation and screening of Pichia pastoris

The recombinant vector pPIC-EGF-n was digested with Bln I to linearize it, and transformed into Pichia pastoris by electroporation. Pick positive transformants. For the methods of electrotransformation and skipping positive transformants, refer to the operation manual of Invitrogen Company.

Use the specific primer of EGF-n gene by the PCR method (5' end gene primer: GAATTCAATTCTTACTCTGAATGTCCA; 3' end vector primer 3'AOX: GCAAATGGCATTCTGACATCC) carry out PCR detection to the positive transformant.

As shown in the PCR result of Figure 1, there is no target gene fragment in the negative transformant, and the target gene fragment EGF-n is contained in the positive transformant, indicating that the present invention has obtained a positive transformant containing the target fragment EGF-n: recombinant Pichia pastoris .

Example 2 Recombinant expression of porcine epidermal growth factor using recombinant Pichia pastoris of the present invention

1. Express

(1) Expression method

Take the recombinant Pichia pastoris constructed in Example 1 and culture it in 5 mL of BMGY medium at 30°C for 48 hours on a shaker, collect the cells by centrifugation, add 1 mL of BMMY methanol induction medium to suspend the cells, and continue to induce culture at 30°C for 96 hours. Sampling and testing at intervals of 12 hours.

BMGY medium: 2% peptone, 1% yeast extract, 100mM potassium phosphate buffer (pH6.0), 1.34% YNB, 4×10 ^-5 % biotin, 1% glycerol;

BMMY induction medium: 2% peptone, 1% yeast extract, 100 mM potassium phosphate buffer (pH6.0), 1.34% YNB, 4×10 ^-5 % biotin, 0.5% methanol.

(2) detection

2.1 SDS-PAGE analysis of expression product epidermal growth factor

Take the culture medium after 24-96 hours of induction, centrifuge to get 20 μL supernatant for Tricine-SDS-PAGE analysis.

The experimental results are shown in Figure 2. The expression of the recombinant epidermal growth factor protein was identified by Tricine-SDS-PAGE. From the SDS-PAGE electrophoresis, it can be seen that the epidermal growth factor expressed by Pichia pastoris has two protein bands. The molecular weight is about 6.5kD, which is equivalent to the theoretical molecular weight (6.5kD) deduced from the amino acid sequence, indicating that recombinant porcine epidermal growth factor has been expressed.

It can also be seen from Figure 2 that after 24 hours of induction, the recombinant porcine epidermal growth factor is significantly expressed, and the expression level gradually increases with time, and the optimal induction time is 72-96 hours.

2.2 Expression level detection

The content of EGF in the culture fluid (the culture fluid after 72 hours of induced expression) was detected by using the porcine EGF detection kit produced by Cloud Clone Company of the United States. For the operation steps, please refer to the kit instructions.

Concentration standard curve is drawn by the standard, the standard curve, as shown in Figure 3.

EGF standard curve (pg/mL) 250 125 62.5 31.75 15.875 _OD450 1.3564 0.7235 0.4386 0.2958 0.1842

Using the standard curve as a reference, ELISA was performed on the culture fluid samples according to the dilution factor to determine the expression level.

The calculation formula is EGF content=(202.92*(A _sample -A _GS115 )-24.66)*dilution factor (100 times)

Through detection, the EGF content of the culture medium was 1.501 μg/mL.

2.3 Activity detection—the effect of recombinant porcine epidermal growth factor (SsEGF) on the growth of fibroblasts

2.3.1 Experimental method

Mouse Balb/c 3T3 cells (purchased from Invitrogen Company) were subpackaged to 96-well plates, ⁵ *10 cells per well, 100ul complete medium, and a carbon dioxide incubator overnight; the cells were replaced with maintenance medium ( Maintenance medium: 4% FBS, 1% Penicillin/Streptomycin, DMEM-high glucose), overnight in a carbon dioxide incubator; prepare samples as shown in Table 1 with maintenance medium, wherein hEGF is human recombinant EGF, purchased from Peprotech, SsEGF Recombinant porcine epidermal growth factor (SsEGF) culture medium (induced expression 72h) of the present invention prepared for the present embodiment; add sample, and set three blank controls, respectively: a. no cells, add maintenance medium; b. cells , add maintenance medium; c. Cells are halved, add maintenance medium, and culture in a carbon dioxide incubator for 72 hours; 5mg/ml MTT is prepared now, use maintenance medium to add 900μL MTT solution according to 9ml medium, prepare solution A, absorb the old culture Base, add 110 μL solution A to each well, and incubate in a carbon dioxide incubator for 4 hours; absorb solution A, add 150 μL DMSO, shake for 5 minutes, measure the absorbance at 490 nm, and compare the absorbance at 570 nm; calculate and draw the growth curve.

Table 1 EGF concentration gradient

Concentration (ng/mL) 1 3 4 hEGF 128 1 0.25 EGF 128 1 0.25

2.3.2 Experimental results

The experimental results are shown in Table 2 and Figures 7-8:

Table 2 Effect of different concentrations of EGF on the growth rate of fibroblasts

Concentration (ng/mL) 128 1 0.25 hEGF 119.7 19.16 25.98 EGF 340.2 76.11 45.14

The experimental results show that the recombinant porcine epidermal growth factor prepared by the inventive method has a significant promoting effect on the growth rate of fibroblasts, and along with the raising of EGF concentration, the growth rate of fibroblasts gradually increases; and, with commercially available Compared with hEGF, the activity of the recombinant porcine epidermal growth factor prepared by the method of the present invention is significantly improved, wherein, at a concentration of 1ng/mL, its activity is 3.97 times that of hEGF, and a completely unexpected technical effect has been achieved. .

2. Separation and purification

Take the culture solution fermented in step 1, centrifuge at 12000rpm at 4°C for 5min to obtain the supernatant and put it into a 3500Da dialysis bag and pass 10 times the volume of buffer [1% CF ₃ COOH, 5% HCOOH (vol/vol), 1% NaCl, 1M HCl] dialyzed overnight at 4°C. All separations were performed at low temperature with a constant flow rate of 1 mL/min and operating parameters controlled by an Altex model 324-40 chromatograph equipped with a 2 mL sample injection loop. Dialyzed proteins were detected by using an LDC Spectromonitor III variable wavelength spectrophotometer. The fragments collected during the preparation process were diluted with sterile water at a ratio of 1:1, which can effectively reduce the concentration of acetonitrile for better hydrophobic interaction. Then 2mL aliquots were transferred to a stainless steel column filled with Ultrasphere-ODS (inner diameter 15cm*4.6mm, and the main solvent was 0.2% heptafluorobutyric acid (C ₃ F ₇ COOH)/H ₂ O in advance, pH2.3 balanced). After loading, the chromatography was performed in primary and secondary solvents (50% acetonitrile/50% H ₂ O/0.2% C ₃ F ₇ COOH, where the volume ratio of acetonitrile to H ₂ O was 50:50, C ₃ H ₇ A linear 50 min gradient was run between COOH acetonitrile and _H2O (0.2% of the total volume) and the eluted material was recovered between 36-40 min. The recovered material was placed in a semi-preparative column of C18 (Waters Company) with an inner diameter of 30cm*9mm in 32.5% acetonitrile/67.5% H ₂ O/0.2% C ₃ H ₇ COOH (wherein the volume ratio of acetonitrile to H ₂ O was 32.5: 67.5, C ₃ H ₇ COOH is 0.2% of the total volume of acetonitrile and H ₂ O) and 40% acetonitrile/60% H ₂ O/0.2% C ₃ H ₇ COOH by a 50-minute linear gradient elution, in this system EFG was eluted at 28.7min to obtain an eluate, the elution diagram is shown in Figure 4, the concentration of EGF in the eluate was 43 μg/mL, and the purity could reach more than 80%.

Experimental result shows, the inventive method can express recombinant porcine epidermal growth factor (SsEGF), and its activity is high, and expression level is also higher, adopts the nutrient solution that engineering bacterium fermentation of the present invention obtains and the eluate after separation and purification The EGF has high activity and high content, is safe and has high nutritional value, and can be used to prepare medicines or feed additives for improving growth performance of piglets.

Embodiment 3 Fermentation tank fermentation of engineering bacteria of the present invention

1. Fermentation method

Get the recombinant Pichia pastoris constructed in Example 1 and ferment in a 50L fermenter. The fermentation of recombinant yeast is high cell density fed-batch fermentation. The fermentation process is divided into strain culture stage, carbon source feeding stage and induced expression stage. For specific methods, please refer to the operation manual of "Pichia Fermentation Process Guidelines", version B053002, pages 1-7

(https://tools.thermofisher.com/content/sfs/manuals/pichiaferm_prot.pdf).

During the induction process, samples were taken every 12 hours to measure the accumulated amount of expressed EGF and Tricine-SDS-PAGE of the expressed protein.

2. Detection

The experimental results showed that: the Pichia recombinant strain P. pastoris EGF-14 with high expression level at the shaker level was selected for 50L fermenter fermentation research.

As shown in Figure 5, no EGF could be detected in the fermentation supernatant during the bacterial growth stage before induction. With the induction of methanol, the EGF content in the supernatant increased significantly, and the protein continued to accumulate. After being induced by methanol for 72h, the EGF content (fermentation titer) reached 5μg/mL.

The experimental results show that the recombinant porcine epidermal growth factor can be efficiently expressed by the method of the present invention, specifically, 250000ug of the recombinant porcine epidermal growth factor can be obtained by fermenting and culturing in a 50L fermenter.

Embodiment 4 Comparison of the method of the present invention and the direct expression of natural porcine epidermal growth factor gene

The EGF and the EGF-n gene fragments of the present invention are cloned into the Pichia pastoris constitutive expression vector pGAPα through the EcoRI/NotI site to obtain the recombinant vectors pGAP-EGF and pGAP-EGF-n. The DNA recombination operation is mainly carried out according to the "Molecular Cloning Experiment Manual".

The recombinant vectors pGAP-EGF and pGAP-EGF-n were digested with Bln I to linearize them, and transformed into Pichia pastoris by electroporation. Pick positive transformants. For electrotransformation and screening methods, see the Invitrogen Company Manual.

Inoculate pGAP-EGF and pGAP-EGF-n positive clones in liquid YPD medium, culture at 30°C, 220rpm for 60 hours, use the trichloroacetic acid method (according to the "Refined Protein Science Experiment Guide") to remove the The protein was precipitated and the expression of both sequences was analyzed by protein electrophoresis.

As shown in Figure 6, adopt the gene expression of natural porcine epidermal growth factor, porcine epidermal growth factor (6.5KD place) expression level is low, and adopt sequence EGF-n expression of the present invention, porcine epidermal growth factor (6.5KD place) expression level substantial improvement.

The experimental results show that the natural porcine epidermal growth factor gene cannot be highly expressed in vitro, but the porcine epidermal growth factor can be efficiently expressed by the gene EGF-n designed in the present invention.

The beneficial effect of the present invention is illustrated below in the mode of experimental example:

Experimental example 1 The influence of recombinant porcine epidermal growth factor (SsEGF) prepared by the method of the present invention on the performance of nursery piglets

By adding the recombinant porcine epidermal growth factor (SsEGF) culture fluid (induced expression 72h) of the present invention prepared in Example 2 and the fermentation broth of the PIC9K vector transformation strain GS115-9K not containing the EGF gene as a control in the nursery piglet diet, To verify the animal use effect of porcine epidermal growth factor.

1. Test materials and methods

1.1 Test animals

This experiment completely followed the random selection of piglets in the nursery stage with different body weights and good eye health and development.

1.2 Test time and place

The test period was from December 29, 2014 to January 31, 2015, a total of 34 days. For the test piglets, No. 1 pen is from 50 days to 84 days; No. 2 to No. 6 pens are from 45 days to 79 days. The test site is a pig farm in Dayi, Sichuan.

1.3 Experimental design

Randomly select 134 weaned piglets, set up 12 treatments, and divide them into test group 1, test group 2, test group 3, test group 4, test group 5 and test group 6. The feed number of the test group 1 was 1, the feed number of the test group 2 was 2, the feed number of the test group 3 was 3, and so on. See Table 1.

Table 1 Experimental design

group Feed number group Sample situation Test group 1 1 11 piglets EGF control Test group 2 2 11 piglets EGF control Test group 3 3 11 piglets EGF Test group 4 4 12 piglets EGF Test group 5 5 11 piglets blank Test group 6 6 12 piglets blank

1.4 Test diet

The formula of the experimental diet was based on the nursery feed of the pig farm, and the EGF sample liquid and the EGF sample liquid control were added to the original nursery feed, and the addition amount was 1‰ respectively.

1.5 Feeding management

The experimental pig house adopts the way of high-bed captive breeding, ad libitum intake of food and water, indoor temperature control at 22-32 ℃, humidity 80%-85%; regular observation and feeding every day (7:00, 11:00, 15:00, 18:00), it is advisable to have a little surplus material in the feeding trough for each feeding; other feeding and immunization procedures should be carried out according to the routine operation procedures of the pig farm; treatment should be as little as possible, and daily injections should not be used for health care.

1.6 Test measurement indicators

Average daily gain (ADG): Weigh on an empty stomach in the morning on the 1st and 34th day of the test respectively, and calculate the ADG in the growth stage;

Average Daily Feed Intake (ADFI): Observe the feed intake of piglets in units of repetitions every day, then calculate the feed consumption in units of repetitions every Sunday morning, and calculate the ADFI of each repetition;

Feed-to-weight ratio (F/G): Calculate F/G based on weight gain and feed intake;

Piglet diarrhea rate: observe and record the diarrhea situation of each pig in repeated units every day before cleaning the pen (8:00 and 14:00 every day), diarrhea frequency = number of piglets with diarrhea / (total number of pigs × time) ×100%;

Mortality rate (%)=number of dead pigs/total number of pigs×100%.

2 Test results

Experimental results show that the recombinant porcine epidermal growth factor (SsEGF) culture solution expressed by the method of the invention can effectively improve the production performance of piglets, and has a significant effect on the weight gain of nursery piglets.

In summary, the present invention has constructed recombinant Pichia containing EGF-n, and adopted the recombinant Pichia expression to obtain highly active recombinant porcine epidermal growth factor (SsEGF), which can effectively promote the growth of fibroblasts and improve Pig production performance, good application prospects.

Claims (10)

1. Nucleotide sequence shown in 1.seq id no.2.
2. 2. a kind of recombinant vector it is characterised in that: it includes the nucleotide sequence shown in seq id no.2；Preferably, described heavy Group carrier is restructured Pichia pastoris in expression carrier ppic9k.
3. 3. a kind of recombinant bacterium it is characterised in that: it includes restructuring described in claim 2 and carries；Preferably, described recombinant bacterium is attached most importance to Group Pichia sp..
4. 4. a kind of method of Prepare restructuring pig's epidermal growth factor it is characterised in that: it is by the restructuring described in claim 3 Bacterium, is placed in culture in microzyme culture medium, and methanol induction is expressed, you can.
5. 5. method according to claim 4 it is characterised in that: it comprises the steps:

   (1) thalli growth: recombinant bacterium is inoculated in bmgy culture medium, cultivates 36～60h in 25～35 DEG C, bacterium is collected by centrifugation Body；

   (2) abduction delivering: use bmmy inducing culture suspension thalline, inducing culture 24～96h at 25～35 DEG C again.
6. 6. method according to claim 5 it is characterised in that: in step (1), cultivation temperature is 30 DEG C, and incubation time is 48h；In step (2), cultivation temperature is 30 DEG C, and induction time is 72h.
7. 7. the expression product that claim 4～6 any one methods described prepares.
8. 8. a kind of method of purification of Recombinant pig's epidermal growth factor it is characterised in that: step is as follows:

   A, take expression product described in claim 7, centrifugation, dialysis；

   B, chromatographic isolation, you can.
9. 9. the purified product that claim 8 methods described prepares.
10. 10. purified product described in expression product, claim 9 described in claim 7 improves the medicine of piglet growth performance in preparation Purposes in thing or feed additive.