Recombinant pinellia ternate agglutinin expression vector, construction method and application of recombinant pinellia ternate agglutinin expression vector

Abstract

The invention provides a recombinant pinellia ternata lectin expression vector, a construction method and application thereof, and belongs to the technical field of biology. According to the invention, RNA (Ribonucleic Acid) of pinellia ternata is successfully extracted, synthesis of a first chain of cDNA (Complementary Deoxyribonucleic Acid) is completed, and nine pinellia ternata lectin genes PTA1-1, PTA1-2, PTA1-3, PTA1-4, PTA2, PTA3, PTA4, PTA5 and PTA6 are successfully cloned. Nine pinellia ternate agglutinin gene promoter fragments are successfully cloned, and a cis-acting element analysis result shows that the pinellia ternate agglutinin gene promoter contains core promoter elements CAAT-box and TATA-box as well as cis-acting elements such as light response, hormone response, stress correlation, plant growth and development regulation and the like, so that a new thought is provided for attenuation of pinellia ternate; and a theoretical basis is provided for medicine development based on the pinellia ternate agglutinin. According to the construction method disclosed by the invention, six pinellia ternate agglutinin expression vectors, namely pET-28a (+)-PTA1-1, pET-28a (+)-PTA1-2, pET-28a (+)-PTA1-3, pET-28a (+)-PTA1-4, pET-28a (+)-PTA5 and pET-28a (+)-PTA6, are successfully constructed.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and particularly relates to a recombinant Pinellia ternata agglutinin expression vector, a construction method and application thereof. BACKGROUND

[0002] Pinellia ternata (Thunb.) Breit is a traditional Chinese medicinal material in China, which is rich in a large amount of active ingredients. Modern pharmacological studies have shown that Pinellia ternata has the effects of relieving cough and asthma, stopping vomiting, anti-tumor, anti-aging, sedation and hypnotic. Pinellia ternata agglutinin (PTA) is a plant protein isolated from Pinellia ternata tubers, which has various biological functions, such as anti-insect, anti-bacterial, anti-viral, anti-tumor effects, and is widely used in agricultural pest control. Pinellia ternata agglutinin is a special component of Pinellia ternata protein, which has both medicinal activity and physiological toxicity. The toxicity includes mucous membrane toxicity, liver and kidney toxicity, and pregnancy toxicity. Calcium oxalate and Pinellia ternata protein are the components of Pinellia ternata toxic needle crystals. Pinellia ternata agglutinin is one of the main components of Pinellia ternata protein, so some scholars believe that Pinellia ternata agglutinin is a toxic component of Pinellia ternata. Pinellia ternata agglutinin can cause cell inflammation by inducing neutrophil migration, activating NF-κB pathway, releasing cytokines, and increasing the production of reactive oxygen species. In addition, Pinellia ternata agglutinin can also induce apoptosis of human tumor Bel-7404 cells. Heating or processing with ginger can reduce the content of Pinellia ternata agglutinin.

[0003] In recent years, researchers have also explored Pinellia ternata agglutinin gene. In 2003, researchers first cloned Pinellia ternata agglutinin gene, which encodes a lectin with secondary structure containing alpha helix and beta fold, and amino acid sequence containing three conserved mannose binding sites. Pinellia ternata agglutinin is a tetramer composed of two N-terminal and C-terminal domains. The molecular weight of Pinellia ternata agglutinin gene is 29 kDa, which can agglutinate rabbit red blood cells, and D-mannose can inhibit its coagulation activity. Pinellia ternata agglutinin has anti-tumor effect, which can inhibit the cell proliferation of mouse malignant sarcoma cells, cervical cancer cells and human chronic myelogenous leukemia cells. In addition, Pinellia ternata agglutinin gene can target methyl bodies to produce cytotoxicity to cancer cells. Pinellia ternata agglutinin transgenic plants have certain resistance to whitefly, aphid, lepidopteran insects, virus and bacteria. Therefore, systematic study of Pinellia ternata agglutinin can not only explore new functions of active ingredients of Pinellia ternata, but also provide ideas for Pinellia ternata attenuation and drug development based on Pinellia ternata agglutinin. SUMMARY

[0004] The present application aims to provide a recombinant Pinellia ternata agglutinin expression vector, a construction method and application thereof. The present application successfully clones the promoter fragment of Pinellia ternata agglutinin gene and constructs the Pinellia ternata agglutinin expression vector, which provides a new idea for Pinellia ternata attenuation and a theoretical basis for drug development based on Pinellia ternata agglutinin.

[0005] In order to achieve the above-mentioned purposes, the present application provides the following technical solutions.

[0006] The present application provides a construction method of a recombinant pinellia pedatisecta agglutinin expression vector, comprising the following steps:

[0007] (1) Extracting pinellia pedatisecta RNA, reverse transcribing into cDNA, using a cloning primer set to clone and purify pinellia pedatisecta cDNA, obtaining pinellia pedatisecta agglutinin related genes PTA1, PTA2, PTA3, PTA4, PTA5 and PTA6;

[0008] (2) Connecting PTA1, PTA2, PTA3, PTA4, PTA5 and PTA6 to a pUCm-T vector, and after being transferred into DH5a competent cells, performing colony verification, selecting colonies for plasmid extraction and enzyme cutting verification screening, and performing sequencing on screened bacterial liquid, obtaining pinellia pedatisecta agglutinin genes PTA1-1, PTA1-2, PTA1-3, PTA1-4, PTA2, PTA3, PTA4, PTA5 and PTA6;

[0009] (3) Using pinellia pedatisecta agglutinin gene promoter primers to obtain pinellia pedatisecta agglutinin gene promoter sequences through a nested PCR reaction, and performing promoter regulatory element analysis;

[0010] (4) Using corresponding primer sets of PTA1, PTA2, PTA3, PTA4, PTA5 and PTA6 genes and EcoR I and Xhol enzymes to perform enzyme cutting on pinellia pedatisecta agglutinin genes, and increasing pinellia pedatisecta agglutinin gene enzyme cutting sites;

[0011] (5) Using restriction endonuclease EcoR I and Xhol to perform double enzyme cutting reactions on pinellia pedatisecta agglutinin genes and a vector pET-28a(+), performing agarose gel electrophoresis on the target fragments and the empty pET-28a(+) plasmid, cutting and recovering the target fragments and the empty pET-28a(+) plasmid, and connecting them to obtain recombinant pinellia pedatisecta agglutinin expression vectors pET-28a(+)-PTA1-1, pET-28a(+)-PTA1-2, pET-28a(+)-PTA1-3, pET-28a(+)-PTA1-4, pET-28a(+)-PTA5 and pET-28a(+)-PTA6;

[0012] (6) Identifying and performing protein expression on the recombinant pinellia pedatisecta agglutinin expression vector.

[0013] The present application also provides a recombinant pinellia pedatisecta agglutinin expression vector constructed by using the construction method of the recombinant pinellia pedatisecta agglutinin expression vector.

[0014] The present application also provides an application of the recombinant pinellia pedatisecta agglutinin expression vector in pinellia pedatisecta research.

[0015] The application also provides a pinellia ternata agglutinin prepared by using the recombinant pinellia ternata agglutinin expression vector.

[0016] The application also provides an application of the pinellia ternata agglutinin in preparing an anti-insect, anti-bacteria, anti-virus or anti-tumor product.

[0017] (1) The application successfully extracts pinellia ternata RNA, completes the synthesis of the first strand of cDNA, and successfully clones nine pinellia ternata agglutinin genes PTA1-1, PTA1-2, PTA1-3, PTA1-4, PTA2, PTA3, PTA4, PTA5 and PTA6. The application also successfully clones nine promoter fragments of pinellia ternata agglutinin genes, and the cis-acting element analysis result shows that the promoter of the pinellia ternata agglutinin gene contains core promoter elements CAAT-box and TATA-box, and cis-acting elements such as light response, hormone response, stress correlation and regulation of plant growth and development, thereby providing a new idea for pinellia ternata attenuation and a theoretical basis for drug development based on pinellia ternata agglutinin.

[0018] (2) The application successfully constructs six pinellia ternata agglutinin expression vectors pET-28a(+)-PTA1-1, pET-28a(+)-PTA1-2, pET-28a(+)-PTA1-3, pET-28a(+)-PTA1-4, pET-28a(+)-PTA5 and pET-28a(+)-PTA6. The correctness is verified by colony PCR and sequencing. The recombinant pinellia ternata agglutinin proteins expressed by the above-mentioned vectors all have coagulation activity, and the coagulation activity of the pinellia ternata agglutinin protein has species specificity, cannot agglutinate human red blood cells, and can agglutinate mouse and rabbit red blood cells. D-mannose can completely inhibit the coagulation activity of the recombinant pinellia ternata agglutinin protein pET-28a(+)-PTA1-2, but cannot inhibit the coagulation activity of other agglutinins, and the minimum inhibition concentration is 25 mM. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0020] Figure 1 It is a pinellia ternata RNA agarose gel electrophoresis graph, wherein a is a pinellia ternata RNA agarose gel electrophoresis graph; b) a pinellia ternata housekeeping gene agarose gel electrophoresis graph, M is DL2000 DNA Marker, and 1-3 are three repeated samples of the housekeeping gene;

[0021] Figure 2 Agarose gel electrophoresis map of pinellia lectin genes in Example 1 of the present application, wherein M is DL2000 DNA Marker, M1 is DNA Marker V II, and a-f are agarose gel electrophoresis maps of pinellia lectin genes PTA1, PTA2, PTA3, PTA4, PTA5 and PTA6, respectively;

[0022] Figure 3 Agarose gel electrophoresis map of colony PCR verification of pinellia lectin genes in Example 1 of the present application, wherein M is DL2000 DNA Marker, 1-25 are single colonies picked from pinellia lectin LB plates, a is agarose gel electrophoresis map of first group of colonies of pinellia lectin gene PTA1, b and d are agarose gel electrophoresis maps of second group of colonies of pinellia lectin gene PTA1, and c, e and h are agarose gel electrophoresis maps of colonies of pinellia lectin genes PTA3, PTA6, PTA2, PTA4 and PTA5, respectively;

[0023] Figure 4 Agarose gel electrophoresis map of pinellia lectin plasmids in Example 1 of the present application, wherein M is DL2000 DNA Marker, M1 is 1 kb DNA Ladder, and 1-25 are plasmids extracted from bacterial liquid of pinellia lectin genes, a and d are agarose gel electrophoresis maps of first group and second group of pinellia lectin PTA1 plasmids, respectively, and b, c, e, f and g are agarose gel electrophoresis maps of pinellia lectin PTA2, PTA3, PTA4, PTA5 and PTA6 plasmids, respectively;

[0024] Figure 5 Agarose gel electrophoresis map of pinellia lectin enzyme digestion products in Example 1 of the present application, wherein M is 1 kb DNA Ladder, 1-25 are enzyme digestion products of pinellia lectin gene plasmids, a and d are agarose gel electrophoresis maps of first group and second group of pinellia lectin PTA1 enzyme digestion verification, respectively, and b, c, e and g are agarose gel electrophoresis maps of pinellia lectin PTA2, PTA3, PTA4, PTA5 and PTA6 enzyme digestion verification, respectively;

[0025] Figure 6Agarose gel electrophoresis diagram of pinellia ternate lectin gene promoter in example 1 of the present application, wherein M is DL2000 DNA Marker, M1 is DL 2000+DNA Marker, 1 is 1st PCR product, 2 is 2nd PCR product, 3 is 3rd PCR product; a-i are agarose gel electrophoresis diagrams of pinellia ternate lectin gene PTA1-1, PTA1-2, PTA1-3, PTA1-4, PTA2, PTA3, PTA4, PTA5, PTA6 promoters respectively;

[0026] Figure 7 Pinellia ternate lectin gene promoter cis-acting element analysis in example 1 of the present application;

[0027] Figure 8 Agarose gel electrophoresis diagram of plasmid pET-28a(+) in example 1 of the present application, wherein M is 1kb DNA Ladder; 1-4 are pET-28a(+) plasmids;

[0028] Figure 9 Agarose gel electrophoresis diagram of gene increased enzyme cutting site in example 1 of the present application, wherein M is DL2000 DNA Marker; a is agarose gel electrophoresis diagram of gene PTA1-1, PTA1-2, PTA1-3, PTA1-4 increased enzyme cutting site; b is agarose gel electrophoresis diagram of gene PTA2, PTA3, PTA4, PTA5, PTA6 after increasing enzyme cutting site;

[0029] Figure 10For electrophoresis chart of example 1 of the present application, A is empty pET-28a(+) of agarose gel electrophoresis chart of double enzyme cutting of target fragment, wherein M1 is 1kb DNA Ladder, M is DL2000 DNA Marker; a is empty pET-28a(+) enzyme cutting agarose gel electrophoresis chart; b is gene PTA2, PTA3, PTA4, PTA5, PTA6 enzyme cutting agarose gel electrophoresis chart; c is gene PTA1-1, PTA1-2, PTA1-3, PTA1-4 enzyme cutting agarose gel electrophoresis chart; B is recombinant pinellia congesta agglutinin pET-28a(+)-Gene Name bacterial colony PCR verification agarose gel electrophoresis chart, wherein M is DL2000 DNA Marker, M1 is DNA Marker V II; 1-20 are single colonies picked from each recombinant pinellia congesta agglutinin pET-28a(+)-Gene Name LB plate; a-f are agarose gel electrophoresis charts of colony PCR verification of recombinant pinellia congesta agglutinin genes pET-28a(+)-PTA1-1, pET-28a(+)-PTA1-2, pET-28a(+)-PTA1-3, pET-28a(+)-PTA1-4, pET-28a(+)-PTA5, pET-28a(+)-PTA6 respectively;

[0030] Figure 11 For agarose gel electrophoresis chart of recombinant pinellia congesta agglutinin pET-28a(+)-Gene Name plasmid in example 1 of the present application, M1 is 1kb DNA Ladder; a is pET-28a(+)-PTA1-1, ET-28a(+)-PTA1-2, pET-28a(+)-PTA1-3, pET-28a(+)-PTA1-4 agarose gel electrophoresis chart; b is pET-28a(+)-PTA5, pET-28a(+)-PTA6 agarose gel electrophoresis chart;

[0031] Figure 12Figure for recombinant pinellin protein in the embodiment of the present application 1, wherein A is SDS-PAGE electrophoretogram of recombinant pinellin protein, M is pre-dye standard protein Marker (10-180 kDa), 1, 2 are empty pET-28a(+) before and after induction, 3, 4 are pET-28a(+)-Gene Name before and after induction, 5 is the precipitate after cell disruption, 6-8 are supernatant of high, medium and low concentration after cell disruption; a-f are solubility verification SDS-PAGE electrophoretogram of recombinant pinellin protein pET-28a(+)-PTA1-1, pET-28a(+)-PTA1-2, pET-28a(+)-PTA1-3, pET-28a(+)-PTA1-4, pET-28a(+)-PTA5, pET-28a(+)-PTA6; B is microscope graph of coagulation activity of recombinant pinellin protein on human blood;

[0032] Figure 13 Figure for coagulation activity of recombinant pinellin protein in the embodiment of the present application, wherein A is mouse blood, B is rabbit blood;

[0033] Figure 14 Figure for coagulation activity inhibition of recombinant pinellin protein, A is D-mannose, B is D-glucose;

[0034] Figure 15 Figure for coagulation activity inhibition of recombinant pinellin protein in the present application, A is sucrose, B is α-D-lactose;

[0035] Figure 16 Figure for coagulation activity inhibition of recombinant pinellin protein, A is D-trehalose, B is L-rhamnose;

[0036] Figure 17 Effect of recombinant pinellin protein in the embodiment of the present application, wherein A is microscope graph of coagulation activity of recombinant pinellin protein on rabbit blood, B-F are coagulation activity inhibition of recombinant pinellin protein by D-mannose, D-glucose, sucrose, α-D-lactose and D-trehalose, respectively;

[0037] Figure 18 Pinellin housekeeping gene PtGAPDH primers and PTA1, PTA2, PTA3, PTA4, PTA5, PTA6 upper and lower stream primers in the embodiment of the present application;

[0038] Figure 19 PTA1-1, PTA1-2, PTA1-3, PTA1-4, PTA2, PTA3, PTA4, PTA5, PTA6 gene cloning primers;

[0039] Figure 20The primers for cloning the promoters of PTA1-1, PTA1-2, PTA1-3, PTA1-4, PTA2, PTA3, PTA4, PTA5 and PTA6 genes. DETAILED DESCRIPTION

[0040] The embodiments of the present application are described in detail below, which are intended to explain the present application and cannot be understood as a limitation of the present application. The specific techniques or conditions not mentioned in the embodiments are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The reagents or instruments not mentioned by the manufacturer are all conventional products that can be obtained by purchase.

[0041] Example 1

[0042] In Example 1 of the present application, the gene of Pinellia ternata was extracted and an expression vector was constructed, and the specific steps are as follows:

[0043] I. Cloning of Pinellia ternata agglutinin gene 1 and extraction of Pinellia ternata RNA by RT-PCR technology: (1) Extraction of Pinellia ternata RNA: In the present application, the EZgene TM Polyphenol plant RNA extraction kit was used to extract the RNA solution of Pinellia ternata according to the instructions. (2) The conventional method was used for RNA agarose gel electrophoresis, and the results are shown in Fig. Figure 1 . Figure 1 The agarose gel electrophoresis of Pinellia ternata RNA showed 28s and 18s bands, and the brightness of 28s band was higher than that of 18s, indicating that the quality of RNA extraction was good. In summary, the extraction result of Pinellia ternata RNA was good and could be used for the synthesis experiment of cDNA first strand.

[0044] 2. Synthesis and verification of Pinellia ternata cDNA first strand: (1) Synthesis of Pinellia ternata cDNA first strand: In the present application, the TIANScript II M-MLV reverse transcriptase kit was used to synthesize the cDNA first strand of Pinellia ternata. (2) The concentration of cDNA first strand was detected by ultraviolet-visible spectrophotometer, and the concentration was detected by using 1 μL RNase-Free Water. When the concentration was ±1 ng / μL, the concentration was detected by using 1 μL cDNA sample. (3) The synthesis of Pinellia ternata housekeeping gene was verified by using the design primer PtGAPDH (see Fig. Figure 18 ). The synthesis of Pinellia ternata housekeeping gene used a 50 μL reaction system (2×TSINGKE MasterMix 25 μL, PtGAPDH-F 1 μL, PtGAPDH-R 1 μL, cDNA 5 μL, sterilized water 18 μL). The PCR reaction program was pre-denaturation at 94℃ for 3 min, (denaturation at 94℃ for 30 s, annealing at 59℃ for 30 s, extension at 72℃ for 1 min) for 36 cycles, final reaction at 72℃ for 5 min, and storage at 4℃. 1% agarose gel electrophoresis.Figure 1 The agarose gel electrophoresis bands of the Pinellia ternate housekeeping genes show that the bands are between 100-250 bp and are bright. In summary, the first-strand cDNA synthesis result of Pinellia ternate is good, and can be used for subsequent cloning of Pinellia ternate lectin genes.

[0045] 3. Cloning and purifying the target genes of Pinellia ternate: The Pinellia ternate lectin genes cloned by the present application are named PTA1, PTA2, PTA3, PTA4, PTA5 and PTA6, respectively, and the upper and lower primers are designed as Gene Name-F and Gene Name-R (see Table 1) respectively. Figure 18 The reaction system for cloning the Pinellia ternate lectin genes is prepared (2×TSINGKE MasterMix 25 μL, Gene Name-F 1 μL, Gene Name-R 1 μL, cDNA 4 μL, sterilized water 19 μL). The PCR reaction procedure is as follows: pre-denaturation at 94℃ for 3 min, 36 cycles of denaturation at 94℃ for 30 s, annealing at X℃ for 30 s, extension at 72℃ for Y min, final reaction at 72℃ for 5 min, and preservation at 4℃. (X and Y are set according to the properties of each gene.) PTA1-F, PTA1-R (X=55℃, Y=1 min); PTA2-F, PTA2-R (X=55℃, Y=1 min); PTA3-F, PTA3-R (X=55℃, Y=1 min); PTA4-F, PTA4-R (X=56℃, Y=1 min); PTA5-F, PTA5-R (X=55℃, Y=1 min); PTA6-F, PTA6-R (X=56℃, Y=1 min).

[0046] 1. The quality of the PCR products is detected by 1% agarose gel electrophoresis, the PCR products are recovered by gel, the target genes are purified, and the concentration of the recovered DNA is detected by a life science ultraviolet-visible spectrophotometer, and the result is shown in Table 2. Figure 2 Figure 2 ​The results showed that the predicted size of gene PTA1 was 810 bp, and agarose gel electrophoresis showed a band between 750-1000 bp. The predicted size of gene PTA2 was 1062 bp, and agarose gel electrophoresis showed a band at 1000 bp. The predicted size of gene PTA3 was 426 bp, and agarose gel electrophoresis showed a band between 300-500 bp. The predicted size of gene PTA4 was 1830 bp, and agarose gel electrophoresis showed a band between 1500-2000 bp. The predicted size of gene PTA5 was 549 bp, and agarose gel electrophoresis showed a band at 500 bp. The predicted size of gene PTA6 was 660 bp, and agarose gel electrophoresis showed a band between 500-750 bp. As can be seen, the agarose gel electrophoresis bands of the Pinellia ternata lectin genes PTA1, PTA2, PTA3, PTA4, PTA5, and PTA6 were all consistent with the predicted size. The obtained PCR products were ligated into a T vector for gene sequence preservation.

[0047] III. Validation of the Pinellia ternata lectin gene: 1. Ligation of the Pinellia ternata lectin gene to the pUCm-T vector: This invention uses a rapid ligation kit for T-vector PCR products for the ligation reaction. 2. DH5α transformation using conventional methods. 3. Colony PCR validation: Single colonies of each gene were picked up with a pipette tip in a horizontal laminar flow double-person clean bench and placed in 10 μL of sterile water. A Pinellia ternata lectin gene colony PCR reaction system was prepared (2×TSINGKE MasterMix 5 μL, Gene Name-F 0.25 μL, Gene Name-R 0.25 μL, bacterial suspension 1 μL, sterile water 3.5 μL) for gene colony PCR validation. PCR reaction program: Pre-denaturation 94℃, 8 min, (denaturation 94℃, 30 s; annealing X℃, 30 s; extension 72℃, Y min) 32 cycles, final reaction 72℃, 5 min, stored at 4℃. (X and Y are set according to the properties of each gene.) The quality of the PCR products was detected by 1% agarose gel electrophoresis, and the results are as follows. Figure 3 As shown, the sequence is... Figure 18 .

[0048] Figure 3 The results showed that the predicted size of the Pinellia ternata lectin gene PTA1 was 810 bp. Colony PCR validation yielded two sets of results. The first set of electrophoresis results showed that PTA1 clones numbered 1-5 had a band at 750 bp, indicating positive clones of the correct size. Figure 3 a). The second set of electrophoresis results showed that PTA1 bands were present in the range of 500-750 bp, indicating a positive clone of the appropriate size. Figure 3 b、 Figure 3 d). The predicted size of PTA2 was 1062 bp, and electrophoresis results showed a band of PTA2 between 1000-2000 bp, indicating a positive clone of the same size.Figure 3 f) PTA3 predicted size is 426bp, electrophoresis results show that PTA3 has a band at 500bp, which is a positive clone with correct size and bright band Figure 3 c) PTA4 predicted size is 1830bp, electrophoresis results show that PTA4 has a band at 2000bp, which is a positive clone with correct size and bright band Figure 3 g) PTA5 predicted size is 549bp, electrophoresis results show that PTA5 has a band between 500-750bp, which is a positive clone with correct size and bright band Figure 3 h) PTA6 predicted size is 660bp, electrophoresis results show that PTA6 has a band at 750bp, which is a positive clone with correct size and bright band Figure 3 e) In summary, single colonies with correct size were selected and added to LB liquid medium, and then cultured at 37℃ overnight, followed by plasmid extraction.

[0049] 4. Plasmid preparation: SanPrep column plasmid DNA extraction kit was used in the present application, and plasmid extraction was performed according to the instructions of the kit, and then 1% agarose gel electrophoresis was used to detect the quality of the plasmid. After electrophoresis, the results were observed by gel imaging system, and the results are shown in Figure 4 . Figure 4 It is shown that the predicted size of PTA1 plasmid is 3583bp, and the plasmid verification results have two groups. The first group of agarose gel electrophoresis results show that the bands of PTA1 No. 1, 2, 4 and 5 are all greater than 2000bp, and the band of No. 5 is slightly high Figure 4 a) The second group of agarose gel electrophoresis results show that the plasmid bands of No. 1, 7, 8, 17, 19 and 21 are between 4000-6000bp, the plasmid bands of No. 2, 4, 16 and 24 are at 4000bp, and the plasmid bands of No. 6, 20, 22, 23 and 25 are at 3000bp Figure 4 d) Therefore, the plasmids of the first group No. 1, 2 and 5 and all the plasmids of the second group were selected for subsequent enzyme digestion verification. The predicted size of PTA3 plasmid is 3201bp, and the agarose gel electrophoresis results show that PTA3 No. 3 and No. 5 have two very faint bands between 3000-4000bp, a band at 1500bp and a bright band, No. 3 has a band between 1500-2000bp and a bright band; No. 7 has two very faint bands between 6000-8000bp and a bright band at 3000bp Figure 4c) Therefore, plasmids numbered 3, 5, 7 were selected for subsequent enzyme digestion verification. The predicted size of the Pinellia pedate agglutinin PTA4 plasmid is 4603 bp, and the agarose gel electrophoresis result shows that PTA4 No. 7 has three very faint bands between 6000-8000, and a bright band between 2500-3000 bp; No. 12-14 has two very faint bands between 3000-4000 bp, and a bright band at 1500 bp Figure 4 e) Therefore, plasmids numbered 7, 12-14 were selected for subsequent enzyme digestion verification. The predicted size of the Pinellia pedate agglutinin PTA5 plasmid is 3322 bp, and the agarose gel electrophoresis result shows that PTA5 No. 3-12, 14, 15 has two very faint bands between 3000-4000 bp, and a bright band at 1500 bp; No. 1 has a very faint band at 2500 bp, and a bright band between 1000-1500 bp; No. 13 has two very faint bands at 4000 bp, and a bright band between 1500-2000 bp Figure 4 f) Therefore, all plasmids were selected for subsequent enzyme digestion verification. The predicted size of the Pinellia pedate agglutinin PTA6 plasmid is 3433 bp, and the agarose gel electrophoresis result shows that PTA6 No. 1 has two very faint bands between 4000-6000 bp, and a bright band at 2000 bp; No. 4 has two very faint bands at 3000 bp, and a bright band between 1000-1500 bp; No. 7 has two very faint bands between 3000-4000 bp, and a bright band at 1500 bp Figure 4 g) Therefore, plasmids numbered 1, 4, 7 were selected for subsequent enzyme digestion verification.

[0050] 5. Enzymatic identification: The Pinellia plasmid was subjected to enzyme digestion reaction with restriction endonuclease EcoR I, Hind III, and a 10 μL reaction system (10 x M Buffer 1 μL, EcoR I 0.25 μL, Hind III 0.25 μL, Pinellia plasmid 4 μL, sterilized water 4.5 μL) was prepared, and the reaction was carried out at 37 °C for 1 h. The plasmid quality was detected by 1% agarose gel electrophoresis, and the results were observed by gel imaging system, as shown in Figure 5 Figure 5 The enzyme digestion verification result of the Pinellia pedate agglutinin PTA1 plasmid has two groups. The first group of agarose gel electrophoresis result shows that PTA1 No. 1, 2, 5 all have a band at 4000 bp, No. 1, 2 have a very faint band at 1000 bp, and No. 5 has a very faint band between 1500-2500 bp Figure 5 a) The second group of agarose gel electrophoresis result shows that PTA1 No. 2 has no band, No. 21 has a band that is too high, No. 1, 4, 6-8, 16, 17, 19, 20, 22-25 have a band between 4000-6000 bp, among which No. 4, 16, 24 have a band between 1000-1500 bp​Figure 5 d) Therefore, the first group of No. 1, 2 bacteria, the second group of No. 4, 16, 24 bacteria are selected and sent to Zhejiang Shangya Biotechnology Co., Ltd. for sequencing. The results of enzyme digestion agarose gel electrophoresis of PTA2 show that PTA2 No. 2, 7, 8, 11 have bands between 4000-5000bp, No. 2 has bands between 1000-1500bp, No. 7 has bands between 500-1000bp Figure 5 b) Therefore, the No. 2, 7 bacteria are selected and sent for sequencing. The results of enzyme digestion agarose gel electrophoresis of PTA3 show that PTA3 No. 3, 5, 7 have bands between 4000-5000bp, No. 3 has bands at 8000bp and 3000bp Figure 5 c) Therefore, the No. 3 bacteria are selected and sent for sequencing. The results of enzyme digestion agarose gel electrophoresis of PTA4 show that PTA4 No. 7, 12-14 have bands between 4000-5000bp, No. 7 has very faint bands between 500-1000bp Figure 5 e) Therefore, the No. 7, 14 bacteria are selected and sent for sequencing. The results of enzyme digestion agarose gel electrophoresis of PTA5 show that PTA5 No. 1 has bands between 2500-3000bp; No. 3, 4, 11-15 have bands between 3000-4000bp, wherein No. 13 has bands between 500-1000bp Figure 5 f) Therefore, the No. 13 bacteria are selected and sent for sequencing. The results of enzyme digestion agarose gel electrophoresis of PTA6 show that PTA6 No. 1, 4, 7 have bands at 4000bp, wherein No. 1 has bands between 500-1000bp Figure 5 g) Therefore, the No. 1 bacteria are selected and sent for sequencing.

[0051] 6, sequencing: the bacteria liquid identified by enzyme digestion is sent to Zhejiang Shangya Biotechnology Co., Ltd. for sequencing, and the pinellia ternata lectin gene sequence is obtained by sequencing. Four homologous genes of PTA1 are obtained by sequencing, which are named as PTA1-1, PTA1-2, PTA1-3 and PTA1-4 respectively. The PTA1-1, PTA1-2, PTA1-3, PTA1-4, PTA2, PTA3, PTA4, PTA5 and PTA6 gene sequences are compared with the predicted CDS sequence, and the results show that the similarity of each pinellia ternata lectin gene sequence is higher than 92%, and the pinellia ternata lectin gene sequence is shown as SEQ ID NO. 1-9. In summary, the sequences obtained by the application are potential pinellia ternata lectin coding genes, which can be used for subsequent research.

[0052] Table 1 similarity comparison table of pinellia ternata lectin gene and predicted CDS sequence

[0053]

[0054] IV. Study on transcriptional regulation of pinellia lectin gene

[0055] 1. Nest PCR reaction to obtain pinellia promoter sequence: The pinellia lectin gene promoter is cloned by chromosome walking technology in the present application. The promoters of the cloned genes PTA1-1, PTA1-2, PTA1-3, PTA1-4, PTA2, PTA3, PTA4, PTA5 and PTA6 are respectively designed with primers Gene Name-SP1, Gene Name-SP2, Gene Name-SP3 (see Figure 20 ), and the primers are subjected to thermal asymmetric PCR reaction with the merging primers AP1, AP2, AP3 and AP4 (purchased) of the kit Genome Walking Kit to clone the promoter sequence.

[0056] (1) The conventional 1st PCR reaction is adopted, and the PCR reaction program is as follows: 94℃ 1min, 98℃ 1min, (94℃ 30s, X℃ 1min, 72℃ 2min) 5 cycles, 94℃ 30s, 25℃ 3min, 72℃ 2min, (94℃ 30s, X℃ 1min, 72℃ 2min, 94℃ 30s, X℃ 1min, 72℃ 2min, 94℃ 30s, 44℃ 1min, 72℃ 2min) 15 cycles, 72℃ 10min, 4℃ preservation. (X is set according to the TM value of each primer) (2) The conventional 2nd PCR reaction is adopted, and the PCR reaction program is as follows: (94℃ 30s, X℃ 1min, 72℃ 2min, 94℃ 30s, X℃ 1min, 72℃ 2min, 94℃ 30s, 44℃ 1min, 72℃ 2min) 15 cycles, 72℃ 10min, 4℃ preservation. (X is set according to the TM value of each primer) (3) The conventional 3rd PCR reaction is adopted, and the reaction program is the same as that of the 2nd PCR reaction. (4) The 1st PCR reaction product, the 2nd PCR reaction product and the 3rd PCR reaction product are detected by 1% agarose gel electrophoresis, and the 3rd PCR reaction product is recovered and sent for sequencing. The agarose gel electrophoresis result is shown in Figure 6 .

[0057] Figure 6 It is shown that the 3rd PCR product of the gene PTA1-1 has a bright band at 750bp ( Figure 6 a). PTA1-2 uses AP1, and the electrophoresis result shows that the 3rd PCR product of the gene PTA1-2 has a bright band at 2000bp ( Figure 6 b). PTA1-3 uses AP2, and the electrophoresis result shows that the 3rd PCR product of the gene PTA1-3 has three bands between 500-750bp, and one of the bands is the brightest.Figure 6 c) PTA1-4 with AP4, electrophoresis results show that the 3rd PCR product of gene PTA1-4 has a bright band between 1000-2000bp Figure 6 d) PTA2 with AP3, electrophoresis results show that the 3rd PCR product of gene PTA2 has a bright band at 1000bp Figure 6 e) PTA3 with AP1, electrophoresis results show that the 3rd PCR product of gene PTA3 has a band at 750bp Figure 6 f) PTA4 with AP1, electrophoresis results show that the 3rd PCR product of gene PTA4 has a bright band between 250-500bp Figure 6 g) PTA5 with AP1, electrophoresis results show that the 3rd PCR product of gene PTA5 has a band at 2000bp Figure 6 h) PTA6 with AP1, electrophoresis results show that the 3rd PCR product of gene PTA6 has a bright band between 1500-2000bp Figure 6 i) In summary, the bright bands of the 3rd PCR products are selected, cut and recovered for sequencing.

[0058] 2, Promoter regulatory element prediction

[0059] The present application predicts the position and number of cis-acting elements of the pinellia agglutinin promoter sequence by online analysis of the pinellia agglutinin promoter sequence through the PlantCARE website, and predicts potential transcription factors. The pinellia agglutinin gene cis-acting element map is drawn by TBtools software, and the results are shown in Figure 7 Through sequencing, 9 pinellia agglutinin gene promoters are obtained, and the sequences upstream of ATG are screened. The sizes of the cloned PTA1-1, PTA1-2, PTA1-3, PTA1-4, PTA2, PTA3, PTA4, PTA5 and PTA6 promoter fragments are 757bp, 1213bp, 393bp, 585bp, 1086bp, 711bp, 335bp, 2126bp and 1493bp (SEQ ID NO. 10-SEQ ID NO. 18) respectively.

[0060] Through analysis, it is known that the pinellia agglutinin gene PTA1-1, PTA1-2, PTA1-3, PTA1-4, PTA2, PTA3, PTA4, PTA5 and PTA6 promoter sequences all contain light response elements, and it is speculated that the expression of pinellia agglutinin gene will be regulated by the light system.

[0061] The pinellia agglutinin gene PTA1-4, PTA2 and PTA6 can regulate plant growth and development.

[0062] Five, construction of expression vector of Pinellia ternate agglutinin: 1, preparation of pET-28a(+) plasmid 50 μL vector pET-28a(+) glycerol bacteria were added to 5 mL LB liquid medium, 5 μL kanamycin, 37℃ oscillation culture overnight (12-16h, 220rpm). Extraction of pET-28a(+) plasmid, detection of plasmid concentration, 1% agarose gel electrophoresis detection of plasmid quality, the results are shown in table 2 and Figure 8 .

[0063] Table 2 plasmid pET-28a(+) concentration

[0064]

[0065] From table 5 and Figure 8 It can be seen that the extracted pET-28a(+) plasmid concentration, high purity. The size of the vector pET-28a(+) is 5369bp, agarose gel electrophoresis results show that the number of 1-4 plasmid in 4000-5000bp between the band bright, at 6000bp has very light band. In summary, pET-28a(+) plasmid extraction success, can be used for subsequent enzyme digestion experiment.

[0066] 2, increase the agglutinin gene Pinellia ternate enzyme cutting site: to the cloned gene PTA1-1, PTA1-2, PTA1-3, PTA1-4, PTA2, PTA3, PTA4, PTA5, PTA6, respectively, design primer B-Gene Name-F, B-Gene Name-R (see Figure 19 ), preparation of reaction system (2xTSINGKE MasterMix 25 μL, B-Gene Name-F 1 μL, B-Gene Name-R 1 μL, cDNA 4 μL, sterilized water 19 μL), PCR reaction, increase EcoRⅠ, Xhol enzyme cutting site. PCR reaction program: pre denaturation 94℃, 3min, (denaturation 94℃, 30s; annealing X℃, 30s; extension 72℃, Ymin) 32 cycles, final reaction 72℃, 5min, 4℃ preservation. (X, Y according to the nature of each gene setting.) Subsequently, the results were detected by 1% agarose gel electrophoresis, the target gene was recovered, and the DNA concentration of the recovered DNA was detected by life science ultraviolet visible spectrophotometer, and the results were shown in table 3 and Figure 9The size of gene PTA1-1, PTA1-3, PTA1-4 is 810 bp, the size of PTA1-2 is 812 bp, the agarose gel electrophoresis result shows that the bands of the four genes are all at 750 bp, and the bands are bright; the size of gene PTA2 is 1062 bp, the agarose gel electrophoresis result shows that there is a band between 750-1000 bp; the size of gene PTA3 is 428 bp, the agarose gel electrophoresis result shows that there is a band between 250-500 bp; the size of gene PTA4 is 1830 bp, the agarose gel electrophoresis result shows that there is a band close to 2000 bp; the size of gene PTA5 is 549 bp, the size of gene PTA6 is 660 bp, the agarose gel electrophoresis result shows that there are bands between 500-750 bp, and the band of gene PTA5 is slightly lower than that of gene PTA6; each gene increases the restriction site electrophoresis has a band, which is recovered. The concentration and purity of the recovered target fragment are high (Table 3). In summary, the gene increases the restriction site successfully, which can be used for subsequent enzyme digestion experiment.

[0067] Table 3 Concentration of recovered target fragment

[0068]

[0069]

[0070] 3, Pinellia pedatisecta lectin target fragment, vector pET-28a(+) double enzyme digestion reaction: the restriction endonuclease EcoR I and Xhol were used for the conventional double enzyme digestion reaction of Pinellia pedatisecta lectin gene and vector pET-28a(+). The target gene was recovered by 1% agarose gel electrophoresis detection, and the DNA concentration of the recovered DNA was detected by life science ultraviolet-visible spectrophotometer, and the results are shown in Table 4. Figure 10 Table 4 DNA concentration of recovered target fragment Figure 10Lane A shows that the size of pET-28a(+) is 5369bp, and the band is bright after agarose gel electrophoresis. The size of gene PTA1-1, PTA1-3 and PTA1-4 is 810bp, and the size of PTA1-2 is 812bp. The result of agarose gel electrophoresis shows that there is a band between 750-1000bp. The size of gene PTA2 is 1062bp, and the result of agarose gel electrophoresis shows that there is a band between 750-1000bp. The size of gene PTA3 is 428bp, and the result of agarose gel electrophoresis shows that there is a band between 250-500bp. The size of gene PTA4 is 1830bp, and the result of agarose gel electrophoresis shows that there is a band between 1000-2000bp. The size of gene PTA5 is 549bp, and the result of agarose gel electrophoresis shows that there is a band at 500bp. The size of gene PTA6 is 660bp, and the result of agarose gel electrophoresis shows that there is a band between 500-750bp. The empty pET-28a(+) and the enzyme digestion products of each gene were recovered by cutting the gel (Table 4).

[0071] Table 4 DNA concentration of empty pET-28a(+) and double enzyme digestion of Pinellia ternate lectin gene

[0072]

[0073] In summary, the target fragments and empty pET-28a(+) were successfully digested by enzymes, which can be used for subsequent ligation experiments.

[0074] 4, Ligation of target fragments and pET-28a(+): 10μL reaction system (2μL of target fragment, 1μL of pET-28a(+), 5μL of 2×Ligation Buffer, 1μL of 50% PEG 4000, 1μL of T4 DNA Ligase), 22℃ for 10min.

[0075] 5, Recombinant Pinellia ternate lectin pET-28a(+)-Gene Name was identified by DH5α transformation (resistance to kanamycin), single colony picking, colony PCR verification, sequencing and extraction of recombinant plasmid.

[0076] (1) Figure 10 In Lane B, colony PCR was used to verify the recombinant Pinellia ternate lectin: the size of PTA1-1 is 810bp, and the result of agarose gel electrophoresis shows that there is a bright band between 500-1000bp, which is a positive clone with correct size. Figure 10 In Lane B, a) the size of PTA1-2 is 812bp, and the result of agarose gel electrophoresis shows that there is a bright band between 500-1000bp, which is a positive clone with correct size. Figure 10B in c). The gene PTA1-4 has a size of 810 bp, and the electrophoretic result shows that there is a band between 500-1000 bp, and the band is bright, which is a positive clone with correct size Figure 10 B in c). The gene PTA1-4 has a size of 810 bp, and the electrophoretic result shows that there is a band between 500-1000 bp, and the band is bright, which is a positive clone with correct size Figure 10 B in d). PTA5 has a predicted size of 549 bp, and the electrophoretic result shows that there is a band between 500-750 bp, and the band is bright and without impurity, which is a positive clone with correct size Figure 10 B in e). PTA6 has a predicted size of 660 bp, and the electrophoretic result shows that there is a band at 750 bp, and the band is bright and without impurity, which is a positive clone with correct size Figure 11 B in f). Based on the above, the above-mentioned positive clones are selected and sent for sequencing. (2) Sequencing: the sequencing result of pET-28a(+)-Gene Name is compared with the sequence of the pinellin gene, and the sequence similarity is 100%. Based on the above, the pinellin gene is successfully connected with the vector pET-28a(+). Figure 11 a). The recombinant plasmid pET-28a(+)-PTA5 has a predicted size of 5884 bp, and the recombinant plasmid pET-28a(+)-PTA6 has a predicted size of 5995 bp, and the electrophoretic band is at 6000 bp, which is correct Figure 12 b). The concentration and purity of the recombinant plasmid are high (Table 5).

[0077] Table 5 Concentration of recombinant pinellin pET-28a(+)-Gene Name plasmid

[0078]

[0079] Based on the above, the present application successfully constructs six recombinant pinellin pET-28a(+)-PTA1-1, pET-28a(+)-PTA1-2, pET-28a(+)-PTA1-3, pET-28a(+)-PTA1-4, pET-28a(+)-PTA5, and pET-28a(+)-PTA6.

[0080] Six, expression and solubility verification of recombinant pinellin protein

[0081] 1. Recombinant Pinellia pedatisecta agglutinin protein BL21(DE3) expression: ① 2 μL empty pET-28a(+) / pET-28a(+)-Gene Name plasmid was added to 100 μL BL21 cells, and placed on ice for 30 min. ② After 82 s of 42 °C water bath heat shock, placed on ice for 4 min. ③ 900 μL LB liquid medium was added in a horizontal laminar flow double purification table, and cultured at 37 °C (40 min, 220 rpm). ④ 180 μL bacterial solution was evenly coated on LB solid medium containing kanamycin. The coated plate was inverted at 37 °C overnight (12-16 h). ⑤ The next day, a piece of bacteria was scraped and added to 5 mL LB medium and 5 μL kanamycin, and cultured at 37 °C overnight (12-16 h, 220 rpm). ⑥ 1 mL bacteria was added to 49 mL LB medium and 50 μL kanamycin, and cultured at 37 °C (1-3 h, 220 rpm). OD600 was measured, and when the OD value was 0.6-0.8, the empty pET-28a(+) / pET-28a(+)-Gene Name pre-induction bacterial solution was collected. ⑦ The strain was preserved with 50% glycerol.

[0082] 2. Recombinant Pinellia pedatisecta agglutinin protein IPTG induction: the bacterial solution with OD600 of 0.6-0.8 was taken, and 50 μL IPTG was added to 50 mL bacterial solution to a final concentration of 210 μM, and induced at 16 °C overnight (12-16 h, 150 rpm). The post-induction bacterial solution of empty pET-28a(+) / pET-28a(+)-Gene Name was collected.

[0083] 3. Solubility verification of recombinant Pinellia pedatisecta agglutinin protein: the recombinant Pinellia pedatisecta agglutinin protein was treated by His-tag protein purification kit type I (ultrasonic lysis) in the present application, and eight sample systems were as follows:

[0084] No. 1: 20 μL empty pET-28a(+) bacteria solution before induction + 5 μL 5x SDS-PAGE loading buffer. No. 2: 20 μL empty pET-28a(+) bacteria solution after induction + 5 μL 5x SDS-PAGE loading buffer. No. 3: 20 μL pET-28a(+)-Gene Name bacteria solution before induction + 5 μL 5x SDS-PAGE loading buffer. No. 4: 20 μL pET-28a(+)-Gene Name bacteria solution after induction + 5 μL 5x SDS-PAGE loading buffer. No. 5: a little bit of the precipitate after bacteria lysis is dipped with a gun head + 20 μL ultrasonic lysis buffer + 5 μL 5x SDS-PAGE loading buffer. No. 6: 20 μL supernatant after bacteria lysis + 5 μL 5x SDS-PAGE loading buffer. No. 7: 15 μL supernatant after bacteria lysis + 5 μL ultrasonic lysis buffer + 5 μL 5x SDS-PAGE loading buffer. No. 8: 10 μL supernatant after bacteria lysis + 10 μL ultrasonic lysis buffer + 5 μL 5x SDS-PAGE loading buffer. Only a small amount of protein can be obtained by traditional extraction of Pinellia ternate corms, and a large amount of Pinellia agglutinin protein with biological activity can be obtained by the prokaryotic expression method. The soluble protein concentrations of Pinellia agglutinin pET-28a(+)-PTA1-1, pET-28a(+)-PTA1-2, pET-28a(+)-PTA1-3, pET-28a(+)-PTA1-4, pET-28a(+)-PTA5 and pET-28a(+)-PTA6 are 25.808 mg / mL, 22.093 mg / mL, 31.994 mg / mL, 37.260 mg / mL, 24.631 mg / mL and 20.358 mg / mL, respectively. There is no obvious difference between the electrophoretic bands of empty pET-28a(+) and the five proteins before and after induction. The sizes of pET-28a(+)-PTA1-1, pET-28a(+)-PTA1-2, pET-28a(+)-PTA1-3, pET-28a(+)-PTA1-4, pET-28a(+)-PTA5 and pET-28a(+)-PTA6 are 33.13 kDa, 33.20 kDa, 33.06 kDa, 33.13 kDa, 25.34 kDa and 26.92 Da, respectively, and the sizes of the electrophoretic bands are consistent. The supernatant and the precipitate of PTA1-1, PTA1-2, PTA1-3, PTA1-4, PTA5 and PTA6 all contain the target protein, proving that the six proteins are neutral proteins. Bioinformatics prediction shows that Pinellia agglutinin PTA1-1, PTA1-2, PTA1-3, PTA1-4 and PTA5 are hydrophilic proteins, and the SDS-PAGE electrophoretic results show that the soluble protein content of the five Pinellia agglutinins is obviously higher than that of the insoluble protein, which is consistent with the prediction results.Bioinformatics predicted that PTA6 is a hydrophobic protein, SDS-PAGE electrophoresis results showed that the protein content of the PTA6 was higher, which was consistent with the prediction. But through low temperature, low concentration of IPTG induction, the soluble protein content of PTA6 was improved. In summary, the protein in the supernatant was selected for subsequent biological property detection. Figure 12 A).

[0085] Seven, the biological properties of recombinant PTA6 were detected. 1. Coagulation activity determination: (1) Preparation of 1% red blood cell suspension. Finger blood was collected from volunteers, and orbital blood was collected from BALB / c nude mice, and ear vein blood was collected from experimental rabbits. 0.5 mL of blood was taken in a centrifuge tube, centrifuged for 5 min (4°C, 3000 rpm), and the supernatant was discarded. The red blood cells were washed with physiological saline and centrifuged for 5 min (4°C, 3000 rpm); repeated 4 times. The 1% red blood cell suspension was diluted with physiological saline and stored at 4°C for standby. (2) Species specificity detection of PTA6 coagulation activity. The initial red blood cell morphology was observed under a microscope. 25 μL of 1% red blood cell suspension was taken, 25 μL of PTA6 protein was added, and it was mixed and placed at room temperature for 2 h. The coagulation results were observed under a microscope. Physiological saline was used as a blank control, empty pET-28a(+) was used as a negative control, and 2.4 mg / mL of natural PTA6 was used as a positive control. Recombinant PTA6 proteins pET-28a(+)-PTA1-1, pET-28a(+)-PTA1-2, pET-28a(+)-PTA1-3, pET-28a(+)-PTA1-4 and pET-28a(+)-PTA5 all had coagulation activity and could agglutinate 1% mouse and rabbit red blood cells, but could not agglutinate human red blood cells. In summary, the coagulation activity of PTA6 has species specificity and cannot agglutinate human red blood cells, but can agglutinate mouse and rabbit red blood cells. Figure 13 B and Figure 17 (3) Coagulation activity determination of PTA6. The coagulation activity of PTA6 protein on rabbit blood was observed. 25 μL of physiological saline was added to holes 1-10, 25 μL of empty pET-28a(+) was added to hole 2, 25 μL of PTA6 protein was added to hole 3, and it was mixed and diluted by 2 times in holes 4-10. Finally, 25 μL of 1% red blood cell suspension was added to each of holes 1-10, mixed and placed at room temperature for 3 h, and the results were observed under a microscope. The agglutination titer of red blood cells was u=2 n , n refers to the minimum PTA6 concentration that can agglutinate red blood cells, "+" represents red blood cell agglutination, "-" represents red blood cell non-agglutination, and the results are as follows Figure 14The coagulation effects of different recombinant pinellia lectins are different. The initial concentration of pET-28a(+)-PTA1-1 lectin is 25.808 mg / mL, and the lectin still has a coagulation effect when the red blood cell agglutination titer u is 25, i.e. the protein concentration is 1.613 mg / mL. The red blood cell agglutination titer u of pET-28a(+)-PTA1-2 is 24, i.e. the protein concentration is 2.762 mg / mL, and the lectin still has a coagulation effect. The red blood cell agglutination titer u of pET-28a(+)-PTA1-3 is 25, i.e. the protein concentration is 1.999 mg / mL, and the lectin still has a coagulation effect. The red blood cell agglutination titer u of pET-28a(+)-PTA1-4 is 26, i.e. the protein concentration is 1.164 mg / mL, and the lectin still has a coagulation effect. The red blood cell agglutination titer u of pET-28a(+)-PTA5 is 24, i.e. the protein concentration is 3.079 mg / mL, and the lectin still has a coagulation effect.

[0086] 2. Sugar inhibition experiment: 200 mmol / L D-mannose, D-glucose, sucrose, α-D-lactose, D-trehalose, L-rhamnose solutions were prepared respectively. Take 96-well "V" bottom blood clotting plate, add 25 μL of sugar solution, 25 μL of empty pET-28a(+) to the 2nd well and mix well. Add 25 μL of sugar solution to the 3rd well, then perform dilution by 2 times in the 4th-10th wells, and add 25 μL of pinellia lectin protein to the 3rd-11th wells respectively and mix well. Finally, add 25 μL of 1% red blood cell suspension to each well, mix well, and stand at room temperature for 2 hours. Observe the results under a microscope, and determine the inhibition effect of various sugars on the coagulation ability of recombinant pinellia lectin protein. (1) D-mannose: After D-mannose treatment, pET-28a(+)-PTA1-1, pET-28a(+)-PTA1-3, pET-28a(+)-PTA1-4, and pET-28a(+)-PTA5 still showed great agglutination effect on 1% rabbit blood, proving that D-mannose has no inhibitory effect on the four. D-mannose can completely inhibit the coagulation activity of pET-28a(+)-PTA1-2, and reducing the concentration of D-mannose can restore the coagulation activity of pET-28a(+)-PTA1-2 protein, and the minimum D-mannose inhibition concentration is 25 mM. PTA1-1, PTA1-2, PTA1-3, and PTA1-4 all contain 3 mannose binding sites, but only the coagulation activity of PTA1-2 is inhibited by mannose. By comparing the PTA1 four homologous sequences, it is found that the amino acid sequence of PTA1-2 has 5 threonine, 3 serine, and 1 arginine. PTA1-2 is predicted to contain 8 threonine sites and 18 serine sites. Studies have shown that reducing serine phosphorylation can promote protein expression. Therefore, it is speculated that PTA1-2 is affected by phosphorylation, the protein expression amount is reduced, and it is more susceptible to mannose to inhibit its coagulation activity Figure 17 A, Figure 14Middle B).

[0087] (2) D-glucose: After D-glucose treatment, pET-28a(+)-PTA1-1, pET-28a(+)-PTA1-4, pET-28a(+)-PTA5 still showed a strong agglutination effect on 1% rabbit blood, proving that D-glucose had no inhibitory effect on the above three. PTA1-2, pET-28a(+)-PTA1-3 can still agglutinate 1% rabbit blood, but the agglutination effect is reduced. It is proved that D-glucose can reduce the coagulation activity of pET-28a(+)-PTA1-2, pET-28a(+)-PTA1-3 Figure 17 Middle B, Figure 15 Middle C).

[0088] (3) Sucrose: After sucrose treatment, pET-28a(+)-PTA1-1, pET-28a(+)-PTA1-4, pET-28a(+)-PTA5 still showed a strong agglutination effect on 1% rabbit blood, proving that sucrose had no inhibitory effect on the above three. Recombinant pinellin protein pET-28a(+)-PTA1-2, pET-28a(+)-PTA1-3 can still agglutinate 1% rabbit blood, but the agglutination effect is reduced, especially pET-28a(+)-PTA1-2. It is proved that sucrose can reduce the coagulation activity of recombinant pinellin protein pET-28a(+)-PTA1-2, pET-28a(+)-PTA1-3 Figure 17 Middle A, Figure 15 Middle D).

[0089] (4) α-D-lactose: After α-D-lactose treatment, pET-28a(+)-PTA1-1, pET-28a(+)-PTA1-2, pET-28a(+)-PTA1-4, pET-28a(+)-PTA5 still showed a strong agglutination effect on 1% rabbit blood, proving that α-D-lactose had no inhibitory effect on the above four proteins. Recombinant pinellin protein pET-28a(+)-PTA1-3 can still agglutinate 1% rabbit blood, but the agglutination effect is reduced. It is proved that α-D-lactose reduces the coagulation activity of pET-28a(+)-PTA1-3 Figure 17 Middle B, Figure 16 Middle E).

[0090] (5) D-trehalose: D-trehalose treatment, pET-28a(+)-PTA1-1, pET-28a(+)-PTA1-4, pET-28a(+)-PTA5 showed great agglutination effect on 1% rabbit blood, D-trehalose had no inhibitory effect on the above three. pET-28a(+)-PTA1-2, pET-28a(+)-PTA1-3, the agglutination effect was reduced. It is proved that D-trehalose can reduce the coagulation activity of pET-28a(+)-PTA1-2, pET-28a(+)-PTA1-3 Figure 17 Middle A, Figure 16 Middle F).

[0091] (6) L-rhamnose: After L-rhamnose treatment, pET-28a(+)-PTA1-1, pET-28a(+)-PTA1-4, pET-28a(+)-PTA5 still showed great agglutination effect on 1% rabbit blood, which proved that L-rhamnose had no inhibitory effect on the above three. pET-28a(+)-PTA1-2, pET-28a(+)-PTA1-3 can agglutinate 1% rabbit blood, but the agglutination effect is reduced. L-rhamnose can reduce the coagulation activity of pET-28a(+)-PTA1-2, pET-28a(+)-PTA1-3 ​ Middle B).

[0092] Table 6 Inhibition of L-rhamnose on the coagulation activity of recombinant pinellia agglutinin protein

[0093]

[0094] The above only describes the preferred embodiments of the present application, it should be noted that for those skilled in the art, without departing from the principles of the present application, can make several improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for constructing a recombinant Pinellia ternata lectin expression vector, characterized in that, Includes the following steps: (1) Extract the RNA from Pinellia ternata, reverse transcribe it into cDNA, and use the cloning primer set to clone and purify the cDNA of Pinellia ternata to obtain the genes PTA1, PTA2, PTA3, PTA4, PTA5, and PTA6 related to Pinellia ternata lectin. (2) PTA1, PTA2, PTA3, PTA4, PTA5, and PTA6 were ligated into the pUCm-T vector, transformed into DH5α competent cells, and colony verification was performed. Colonies were selected for plasmid extraction and enzyme digestion verification. The selected bacterial solutions were sequenced to obtain the Banxia lectin genes PTA1-1, PTA1-2, PTA1-3, PTA1-4, PTA2, PTA3, PTA4, PTA5, and PTA6. (3) Using the promoter primers of the Pinellia ternata lectin gene, the promoter sequence of the Pinellia ternata lectin gene was obtained by nested PCR reaction, and the promoter regulatory elements were analyzed. (4) Use the corresponding primer sets of PTA1, PTA2, PTA3, PTA4, PTA5, and PTA6 genes, as well as EcoRI and Xhol enzymes to digest the Pinellia ternata lectin gene to increase the number of enzyme digestion sites of the Pinellia ternata lectin gene. (5) The Banxia lectin gene and vector pET-28a(+) were double-digested using restriction endonucleases EcoRI and Xhol. After agarose gel electrophoresis, the target fragment and empty vector pET-28a(+) plasmid were excised and recovered, and ligated to obtain recombinant Banxia lectin expression vectors pET-28a(+)-PTA1-1, pET-28a(+)-PTA1-2, pET-28a(+)-PTA1-3, pET-28a(+)-PTA1-4, pET-28a(+)-PTA5, and pET-28a(+)-PTA6. (6) Identification and protein expression of the recombinant Pinellia ternata lectin expression vector.

2. The method for constructing the recombinant Pinellia ternata lectin expression vector according to claim 1, characterized in that, In step (2), the gene sequences of the Pinellia ternata lectin genes PTA1-1, PTA1-2, PTA1-3, PTA1-4, PTA2, PTA3, PTA4, PTA5, and PTA6 are shown in SEQ ID NO.1 to SEQ ID NO.

9.

3. The method for constructing the recombinant Pinellia ternata lectin expression vector according to claim 1, characterized in that, In step (3), the promoter sequence of the Pinellia ternata lectin gene is shown in SEQ ID NO.10 to SEQ ID NO.

18.

4. A recombinant Pinellia ternata lectin expression vector constructed using the construction method of the recombinant Pinellia ternata lectin expression vector according to any one of claims 1 to 3.

5. The application of the recombinant Pinellia ternata lectin expression vector as described in claim 4 in the study of Pinellia ternata.

6. A Pinellia lectin prepared using the recombinant Pinellia lectin expression vector of claim 4.

7. The use of the Pinellia ternata agglutinin of claim 6 in the preparation of insect-resistant, antibacterial, antiviral or antitumor products.

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