Recombinant expression and purification method of ampullaria gigas aggregation pheromone protein

Abstract

The invention discloses a recombinant expression and purification method of ampullaria gigas aggregation pheromone protein, and belongs to the technical field of gene engineering. The invention provides a high-efficiency expression method of pomacea canaliculata pheromone protein, which comprises the following steps: connecting a coding gene to a recombinant vector, carrying out small-amount expression induction by using a pichia pastoris expression system or an escherichia coli cell-free expression system, screening a recombinant expression system with positive small-amount expression detection by using SDS-PAGE and WB technologies, and carrying out enlarged fermentation culture, so as to obtain the pomacea canaliculata pheromone protein. And collecting protein, and purifying. Experimental results show that the protein expressed according to the method provided by the invention has the purity of 85% or above, the final concentration of the expressed protein is 0.3 mg / mL, efficient preparation of the high-purity pomacea canaliculata pheromone protein can be realized, and a new technical support is provided for biological control of pomacea canaliculata invasion.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to a method for recombinant expression and purification of Pomacea canaliculata aggregation pheromone protein. Background Art

[0002] The golden apple snail [Pomacea canaliculata (Lamarck, 1819)] belongs to the phylum Mollusca, the class Gastropoda, the order Mesogastropoda, the family Ampullariidae, the genus Pomacea. It is native to the Amazon River Basin in South America and is the only freshwater snail listed as one of the world's 100 malignant alien species. It has a 40-year history of invasion in my country. Due to its fast growth, high reproduction, large appetite, miscellaneous diet, strong resistance to adversity, poor management and lack of natural enemies, it has invaded more than 260 counties (districts) in 17 provinces (municipalities) in my country, seriously harming more than 30 million mu of farmland. The golden apple snail not only causes serious damage and huge economic losses to crop production, aquatic ecosystems and biodiversity, but can also spread zoonotic parasites, threatening human life and health. In order to prevent and control the spread of golden apple snails, domestic and foreign scholars have conducted a lot of research in agricultural control, physical control, chemical control and biological control. However, agricultural and physical control are time-consuming and labor-intensive, and the inefficiency of using large amounts of chemical agents will induce golden apple snails to develop drug resistance, kill non-target beneficial organisms by mistake, and cause environmental pollution and ecological damage. Therefore, it is urgent to develop economically efficient, environmentally friendly and sustainable green control technologies.

[0003] Biological control is a method of controlling golden apple snails by using natural enemies or biological agents. Natural enemy control is to control the number of golden apple snails by using predators and pathogenic microorganisms, while biological agents use products from natural sources (such as plant extracts, pheromones, etc.) to disrupt the reproductive behavior of golden apple snails or inhibit their activity, thereby controlling golden apple snails. Based on the behavioral and biological characteristics of golden apple snails, finding active ingredients or natural substances that have an attracting and aggregation effect on them is a current research hotspot.

[0004] Pheromones are a general term for chemical substances with communication functions. Using the attractant effect of biological pheromones for ecological prevention and control is an important measure for integrated pest control, which is safe, specific and efficient. The vision of the golden apple snail is degraded, and it relies on chemical signals in the secreted body fluids to perceive information, identify directions and track its own kind. There is an obvious aggregation phenomenon within the population. However, in the prevention and control of golden apple snails, the research on pheromones is still in its infancy, and there is still a need to find more substances that interfere with the behavior of golden apple snails and study their use methods. Therefore, it is urgent to provide a recombinant expression and purification method of golden apple snail pheromone protein to efficiently separate the golden apple snail pheromone protein and provide new technical means for the biological control of golden apple snails. Summary of the invention

[0005] The purpose of the present invention is to provide a recombinant expression and purification method of Pomacea canaliculata aggregation pheromone protein to solve the problems existing in the above-mentioned prior art. According to the recombinant expression and purification method provided by the present invention, high-purity Pomacea canaliculata aggregation pheromone protein can be efficiently prepared.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for preparing a Pomacea canaliculata aggregation pheromone protein, comprising the following steps:

[0008] Connecting the coding gene of the aggregation pheromone protein of Pomacea canaliculata to a vector to obtain a recombinant vector;

[0009] Transforming the recombinant vector into an expression system to obtain a recombinant expression system;

[0010] Inducing the recombinant expression system to perform small-scale expression detection;

[0011] The recombinant expression system that is positive in a small amount of expression detection is subjected to amplified fermentation, purification, and protein detection to obtain the Pomacea canaliculata aggregation pheromone protein.

[0012] Preferably, the nucleotide sequence of the gene encoding the Pomacea canaliculata pheromone protein is as shown in SEQ ID NO.2 or SEQ ID NO.4.

[0013] Preferably, the expression system is a Pichia pastoris expression system or an Escherichia coli cell-free expression system.

[0014] Preferably, when the expression system is a Pichia pastoris expression system, the Pichia pastoris expression system is an X33 strain, the vector is a pPICZαA vector, and the transformation method is electrotransformation.

[0015] Preferably, the E. coli cell-free expression system comprises a mixed E. coli extract, a feed buffer, a supplement buffer, amino acids, T7 RNA polymerase and the recombinant vector.

[0016] Preferably, when the expression system is an Escherichia coli cell-free expression system, the vector is a pET23b vector.

[0017] Preferably, the purification method is nickel column purification.

[0018] Preferably, the protein eluent for nickel column purification is 1*PBS containing 20-50 mM imidazole or PBS containing 0.05% polyoxyethylene stearate and 500 mM imidazole.

[0019] The present invention discloses the following technical effects:

[0020] The present invention provides a method for efficiently expressing pheromone protein of Pomacea canaliculata, comprising connecting a coding gene to a recombinant vector, using a Pichia pastoris expression system or an Escherichia coli cell-free expression system for small-scale expression induction, using SDS-PAGE and WB technology to screen the recombinant expression system with positive small-scale expression detection for expanded fermentation culture, collecting protein, and purifying. Experimental results show that the protein expressed according to the method provided by the present invention has a purity of more than 85%, and the final concentration of the expressed protein is 0.3 mg / mL, which can achieve efficient preparation of high-purity Pomacea canaliculata pheromone protein, not only providing a scientific basis for specific trapping of Pomacea canaliculata, but also providing technical support for biological control of Pomacea canaliculata invasion. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 The SDS-PAGE electrophoresis diagram of the small-scale expression experiment; M is a marker, and the molecular weights of the proteins in each band are 14.4, 18.4, 25, 35, 45, 66.2, and 116 (kDa), respectively; 1 is the precipitate, and the arrow marks the location of the target protein band; 2 is the supernatant;

[0023] Figure 2 The figure is the SDS-PAGE electrophoresis diagram of the cell-free expression experiment; wherein, M is a marker, and the molecular weights of the proteins in each band are 14.4, 18.4, 25, 35, 45, 66.2, and 116 (kDa), respectively; 1 is loading; 2 is penetration; 3 is PBS, 0.05% Brij78, pH=7.4, 60mM imidazole elution; 4 is PBS, 0.05% Brij78, pH=7.4, 500mM imidazole elution, and the arrows mark the locations of the target protein bands;

[0024] Figure 3 This is the SDS-PAGE electrophoresis diagram of the protein control experiment; where M is the marker; 1 is the purified target protein;

[0025] Figure 4 This is the agarose gel electrophoresis diagram of the plasmid linearization experiment; where M is the marker; 1 is the non-linearized plasmid; 2 is the linearized plasmid;

[0026] Figure 5 It is the agarose gel electrophoresis diagram of PCR detection experiment; M is Marker; 1-10 are different transformants; Yang is positive control;

[0027] Figure 6 This is the SDS-PAGE electrophoresis diagram of the secretory expression test; M is a marker; 1-7 are different positive transformants; Yin is a negative control; Tris Glycine is a tris-glycine buffer;

[0028] Figure 7 The figure is the WB detection result of the secretory expression test experiment; M is a marker; 1-7 are different positive transformants; Tris Glycine is a tris-glycine buffer;

[0029] Figure 8 The figure is the SDS-PAGE electrophoresis diagram of the purification experiment; wherein, M is the marker; FT is the flow-through solution; W is the washing solution; 20mM (1) and (2) are 1*PBS containing 20mM imidazole; 50mM is 1*PBS containing 50mM imidazole; 500mM is 1*PBS containing 500mM imidazole;

[0030] Fig. 9 It is the SDS-PAGE electrophoresis diagram of the concentrated protein; wherein, M is the marker; and 1 is the concentrated protein. DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] Example 1

[0037] 1. Experimental Materials and Procedures

[0038] 1.1 Components of the E. coli cell-free expression system

[0039] 1.1.1 Optimized Escherichia coli extract (Zubay, 1973) is used to improve the stability of the template during transcription and translation and increase protein production.

[0040] 1.1.2 Optimized reaction buffer containing an energy regeneration system (Kim et al., 1996; Lesley et al., 1991; Pratt, 1984) to provide energy for protein synthesis.

[0041] 1.1.3 Optimized feeding buffers containing salts and other substrates (Kim and Swartz, 1999) to replenish components that are depleted or degraded during protein synthesis, thereby improving the yield of recombinant protein.

[0042] 1.1.4 Amino acids.

[0043] 1.1.5 A mixed enzyme containing T7 RNA polymerase (Studier et al., 1990), suitable for DNA or RNA templates based on T7 promoter.

[0044] 1.2 Plasmid construction

[0045] The amino acid sequence of the recombinant protein of this example is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding the recombinant protein is shown in SEQ ID NO.2.

[0046] SEQ ID NO.1:

[0047] MLYNVTVCTLLLTFSQEDFRRRMASLTVLASILLVCLLPLALPFPTFQDSIPNGKNVVNPC DKSPWPGVGHQASQGGGPRNPFGLDFAAAGNAWTTALCQKDSDGDGVSNGEELGDSSCQ WNATNPVNLASPKGHPGICEPVGSAKCGNSWFACPS.

[0048] SEQ ID NO.2:

[0049] ATGCTATATAACGTTACGGTCTGTACGCTGCTGACATTCTCCCAGGAAGACTTCAGACGTAGAATGGCCTCCCTCACTGTGCTCGCCTCGATCTTGCTGGTGTGTCTCCTACCCTTGGCACTGCCCTTCCCCACCTTCCAGGATAGTATTCCCAATGGTAAGAACGTGGTCAACCCGTGTGACAAGTCTCCTTGGCCTGGTGTTGGTCACCAAGCGAGCCAGGGCGGTGGAC CAAGAAACCCTTTCGGACTTGATTTTGCTGCAGCCGGTAATGCGTGGACGACTGCCCTGTGCCAGAAGGACTCCGATGGTGACGGTGTGTCCAACGGTGAGGAGCTGGGCGACAGCAGCTGCCAGTGGAACGCCACCAACCCCGTCAACCTCGCCAGTCCTAAAGGACATCCAGGTATCTGTGAGCCTGTTGGATCAGCTAAATGTGGAAACAGCTGGTTTGCTTGCCCTTC.

[0050] The pET23b vector was constructed, and the coding gene shown in SEQ ID NO.2 was inserted into the Nde I-Xho I restriction site; the tag was N-terminal 10×His-tagged, the molecular weight of the fusion protein was 17.8 kDa, and the plasmid was extracted using the Progema kit for use.

[0051] 1.3 Cell-free expression

[0052] The following steps are involved:

[0053] The plasmid obtained in 1.3.1 can be directly used in the in vitro transcription and translation system.

[0054] 1.3.2 Mix the E. coli extract, feed buffer, amino acids, T7 RNA polymerase and plasmid, add the mixed reaction solution into the dialysis box, and then immerse the dialysis box in the feed buffer.

[0055] 1.3. Dialyze overnight at 330℃.

[0056] 1.3.4 Take the reaction solution, centrifuge it at 4℃, 12000rpm for 10min, separate the supernatant and precipitate, and detect it by SDS-PAGE.

[0057] 1.4 Cell-free purification

[0058] The following steps are involved:

[0059] 1.4.1 Precipitation The precipitate was dissolved with 1% LMPG, and the supernatant was obtained by centrifugation and incubated with 2 mL of nickel column material (Qiagen, NTAAgarose) overnight (4°C), and purified on a gravity column.

[0060] 1.4.2 Use detergent and imidazole to elute the column. Collect the protein eluate and perform ultrafiltration.

[0061] Protein dissolution buffer A: PBS, 1% LMPG, pH = 7.4;

[0062] Protein incubation Buffer B: PBS, 0.05% Brij78 (polyoxyethylene stearate, as detergent), pH = 7.4;

[0063] Protein elution buffer: PBS, 0.05% Brij78, pH=7.4, different concentrations of imidazole.

[0064] 1.4.3 Take 10 μL of protein sample respectively and perform SDS-PAGE detection.

[0065] 2. Experimental results

[0066] 2.1 Cell-free small-scale expression and scale-up

[0067] After the reaction is completed, the reaction solution is centrifuged at 4°C, 12000 rpm for 10 min. The supernatant and precipitate are taken for SDS-PAGE detection. Figure 1 As shown; Figure 1 The lanes in the middle are proteins that are irrelevant to the experiment and should be ignored. From the results of the small test, it can be seen that the target protein (17.8kDa) was successfully expressed, and the reaction system was directly expanded for purification.

[0068] 2.2 Cell-free expression and purification

[0069] The results are as follows Figure 2 As shown, 500 mM imidazole was eluted and the target protein (17.8 kDa) was successfully separated.

[0070] 2.3 Protein control

[0071] The results are as follows Figure 3 As shown, the target protein band is clear and the size and position do not change.

[0072] 3. Experimental results analysis

[0073] According to the results of the small test, the target protein was successfully expressed. After amplification, it was purified by nickel column and eluted with detergent 0.05% Brij78 + imidazole in sequence. The eluents with different gradients were collected, and the target protein was obtained in PBS, 0.05% Brij78, pH=7.4, 500mM imidazole elution fractions. The eluted samples were concentrated and sent for quality control. The electrophoresis size was 19kDa, which was slightly deviated from the theoretical size of 17.8kDa (it may be slightly larger due to gel running or the properties of the protein itself).

[0074] The protein concentration was detected by SDS-PAGE Coomassie Brilliant Blue staining and BCA method, and the purity was evaluated using Bandscan software. The purity was ≥85%. The protein was preserved in the form of Lyophilized from PBS, 0.05% Brij78, 6% trehalose, pH = 7.4.

[0075] (Total amount 20μg)

[0076] Example 2

[0077] 1. Main Materials and Reagents

[0078] Table 1 Materials and reagents information

[0079]

[0080] 2. Experimental Procedure

[0081] 2.1 Plasmid construction

[0082] 2.1.1 Sequence information

[0083] The amino acid sequence of the recombinant protein of this example is shown in SEQ ID NO.3, and the nucleotide sequence of the gene encoding the recombinant protein is shown in SEQ ID NO.4.

[0084] SEQ ID NO.3:

[0085] MASITVLTSILLVCLLPLAEPFPYFQNRIPNGRNVLNPCDKSAWPGVGHKVSQGGGQR NPFGLDFAAAGYVWTTELCYKDSDGDGQSNGVELGDSMCQWNATNPIPLSRPKGHPGICE PVGSDRCQDSWFSCPPKS.

[0086] SEQ ID NO.4:

[0087] ATGGCCTCCATCACTGTGCTCACCTCGATCTTGCTGGTGTGTCTGCTACCCTTGGCAGAGCCCTTCCCCTACTTCCAGAATAGAATTCCCAACGGTAGGAATGTGCTCAACCCATGTGACAAGTCTGCTTGGCCTGGTGTTGGTCACAAAGTGAGCCAGGGCGGCGGACAAAGAAACCCTTTCGGACTTGATTTTGCTGCAGCC GGTTATGTGTGGACGACTGAACTGTGCTATAAGGACTCCGATGGTGACGGTCAGTCCAACGGCGTGGAGCTGGGCGACAGCATGTGCCAGTGGAACGCCACCAACCCCATCCCCCTCTCCAGACCCAAAGGACATCCAGGTATCTGTGAGCCTGTTGGATCAGATAGATGTCAAGACAGTTGGTTTAGTTGCCCTCCAAAGTCT.

[0088] 2.1.2 Plasmid construction method

[0089] 1) Amplify and extract the vector plasmid containing the target gene

[0090] 2) Subcloned into the eukaryotic expression vector pPICZαA;

[0091] 3) Sequencing to verify the accuracy of the constructed plasmid;

[0092] 4) Obtain the recombinant plasmid containing the target gene using a plasmid extraction kit.

[0093] 2.2 Electroporation and PCR Verification

[0094] 1) Linearization: The recombinant plasmid was digested with Sac1 enzyme overnight and recovered, and the linearized and non-linearized plasmids were detected by electrophoresis;

[0095] 2) Electroporation: Prepare X33 yeast competent cells, electroporate the linearized plasmid into competent cells after 1500V voltage stimulation, spread on YPD solid plates containing bleomycin antibiotics, and culture for 2-3 days;

[0096] 3) Bacterial liquid PCR: Select several single colonies and culture them overnight in YPD liquid medium. Take the bacterial liquid and use the universal yeast primer AOX-1 (primer sequence is as follows) for PCR verification, and use the linearized recombinant plasmid as the positive control.

[0097] AOX-1-F: GACTGGTTCCAATTGACAAGC (SEQ ID NO.5);

[0098] AOX-1-R: GCAAATGGCATTCTGACATCC (SEQ ID NO. 6).

[0099] 2.3 Small-scale secretory expression test

[0100] 1) Inoculation: Select the strains that need to be expressed in the test according to the PCR gel image, and inoculate the strains in the YPD medium in 2.2 (including the X33 / pPICZαA empty vector) into the BMGY medium.

[0101] 2) Induction: Centrifuge the BMGY culture in the previous step, discard the supernatant, replace with BMMY medium, add 0.5% methanol every 12 hours to induce expression, and harvest the bacteria after 72 hours;

[0102] 3) Detection: After the shake flask fermentation, samples were taken for SDS-PAGE and WB verification.

[0103] 2.4 Secretory expression

[0104] 1) Activation of bacterial strains: inoculate 200 μL of positive strains with good expression activity into 50 mL YPD liquid medium for overnight culture;

[0105] 2) Expanded culture: Inoculate 10 mL of the activated bacterial solution in 1 L BMGY medium and wait until the OD 600 After 2-3, centrifuge at 4500rpm for 30min and discard the supernatant;

[0106] 3) Inducing expression: Replace 1 L BMMY medium, add 0.5% methanol every 12 hours to induce expression, collect the bacteria after 72 hours, centrifuge at 4500 rpm for 30 minutes, and retain the supernatant;

[0107] 4) Column material binding: Take 1 / 4 column volume of Ni-NTA filler, balance the column with 2 column volumes of 1*PBS, mix the balanced column material and dialyzed protein supernatant evenly, and incubate at 14°C, 150 rpm for 2 hours.

[0108] 5) Protein elution: gradually add the mixture in the previous step to the binding column. After the supernatant has completely flowed out, add 2 column volumes of 1*PBS to equilibrate the column. Sequentially use 50 mL of 1*PBS containing 20 mM, 50 mM and 500 mM imidazole to elute the target protein. Keep a sample for SDS-PAGE electrophoresis.

[0109] 6) Concentration: The eluted target protein is transferred to a dialysis bag for overnight dialyzation and concentrated with polyethylene glycol for storage.

[0110] 3. Experimental results

[0111] 3.1 Plasmid linearization results

[0112] The results are as follows Figure 4 As shown, after the recombinant plasmid was digested with Sac1, the theoretical value of the linearized fragment was 3973 bp (vector 3593 bp + target gene 380 bp).

[0113] 3.2 PCR test results

[0114] The results are as follows Figure 5 As shown, the target gene size is 380bp plus the sequence on the vector is about 546bp, totaling 926bp. Bands consistent with the positive control appeared in lanes 1-10. We selected transformants 1-7 to arrange expression detection experiments.

[0115] 3.3 Results of secretory expression test

[0116] The results are as follows Figure 6 As shown, the theoretical size of the target protein is about 13.71 kDa. According to the above test results compared with the negative control, the positive transformants showed suspected bands near the size of the target protein, and further WB detection was arranged.

[0117] The results are as follows Figure 7 Specific bands were detected in each transformant lane near the theoretical molecular weight; transformant No. 7 was selected for amplification and purification.

[0118] 3.4 Purification test

[0119] The results are as follows Figure 8 and Fig. 9 As shown, the purified protein was dialyzed and concentrated, and the concentration was about 0.3 mg / mL as measured by BCA.

[0120] 4. Experimental Summary

[0121] In summary, after the target gene was secreted and expressed by the Pichia pastoris expression system, a certain amount of target protein was obtained; the final protein concentration was about 0.3 mg / mL, the volume was 4 mL, the buffer was PBS, and the pH was 7.4.

[0122] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a Pomacea canaliculata aggregation pheromone protein, characterized in that: The following steps are involved: Connecting the coding gene of the aggregation pheromone protein of Pomacea canaliculata to a vector to obtain a recombinant vector; Transforming the recombinant vector into an expression system to obtain a recombinant expression system; Inducing the recombinant expression system to perform small-scale expression detection; The recombinant expression system that is positive in a small amount of expression detection is subjected to amplified fermentation, purification, and protein detection to obtain the Pomacea canaliculata aggregation pheromone protein.

2. The preparation method according to claim 1, characterized in that The nucleotide sequence of the gene encoding the pheromone protein of Pomacea canaliculata is shown as SEQ ID NO.2 or SEQ ID NO.

4.

3. The preparation method according to claim 1, characterized in that: The expression system is a Pichia pastoris expression system or an Escherichia coli cell-free expression system.

4. The preparation method according to claim 3, characterized in that: When the expression system is a Pichia pastoris expression system, the Pichia pastoris expression system is an X33 strain, the vector is a pPICZαA vector, and the transformation method is electrotransformation.

5. The preparation method according to claim 3, characterized in that: The Escherichia coli cell-free expression system comprises a mixed Escherichia coli extract, a feed buffer, a supplement buffer, amino acids, T7 RNA polymerase and the recombinant vector.

6. The preparation method according to claim 3, characterized in that: When the expression system is an Escherichia coli cell-free expression system, the vector is a pET23b vector.

7. The method according to claim 1, characterized in that The purification method is nickel column purification.

8. The method according to claim 7, characterized in that The protein eluent for nickel column purification is 1*PBS containing 20-50mM imidazole or PBS containing 0.05% polyoxyethylene stearate and 500mM imidazole.

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