Homologous recombinant expression vector for expressing Pt5-1c active peptide in dunaliella salina

Abstract

The invention provides a homologous recombinant expression vector for expressing a Pt5-1c active peptide in dunaliella salina, the provided expression vector can efficiently express an exogenous gene in dunaliella salina, and the homologous recombinant expression vector comprises a construction component which comprises a promoter with a nucleotide sequence of SEQ ID NO: 1. According to the constructed expression vector, an exogenous gene is separated from other genes through a P2A sequence, so that the exogenous gene is independently expressed, and interference among the genes is avoided; an iron oxidase signal recognition sequence is introduced while an exogenous gene is inserted, so that active peptides such as Pt5-1c and the like can be guided to be secreted out of cells, and the problems of impurity release, active peptide structure damage and the like caused by obtaining the active peptides by breaking the cells are avoided. According to the constructed expression vector, an enterokinase enzyme cutting site is introduced, so that the active peptide can be conveniently cut and separated in the subsequent separation and purification process, the consumption of reagents and consumables is reduced, the purification efficiency is improved, and the purification cost is reduced.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological genetic engineering, and particularly relates to a homologous recombination expression vector for expressing a Pt5-1c active peptide in Dunaliella salina. BACKGROUND

[0002] Dunaliella salina is a representative eukaryotic microalgae, and its unique biological characteristics make it have great application potential in the field of biological genetic engineering. It can survive in an extreme environment with a salt concentration as high as saturation. This outstanding salt tolerance not only makes it less susceptible to contamination by foreign bacteria during cultivation, reducing the risk of interference with the cultivation system, but also reduces the strict requirements for the cultivation environment. At the same time, the cultivation of Dunaliella salina does not require complex equipment and expensive nutrients, and the cultivation cost is relatively low. It can achieve rapid proliferation in an open or closed large-scale cultivation system, and has a natural advantage in large-scale cultivation. These characteristics make it an ideal expression host for producing exogenous proteins, active peptides and other bioactive substances, and it has broad application prospects in the fields of medicine and health care products.

[0003] However, in the application of Dunaliella salina for expressing active peptides such as Pt5-1c, the existing transgenic system has insurmountable shortcomings. Active peptides usually have the characteristics of small molecular weight and high biological activity, but their accumulation in cells not only may have toxic effects on host cells, but also increases the difficulty of subsequent separation and extraction. The current system lacks an effective mechanism to guide the secretion of active peptides to the extracellular, resulting in a large amount of active peptides remaining in the cells. To obtain these intracellular active peptides, cell disruption and other methods are often required, which not only releases a large amount of impurities such as nucleic acids, proteins, polysaccharides and other impurities in the cells, but also may damage the structure of active peptides and affect their biological activity.

[0004] At the same time, the existing system also lacks convenient separation and purification means. Because the physicochemical properties of active peptides and other impurities in the cells are similar, traditional separation and purification methods such as chromatography and electrophoresis are not only tedious and time-consuming, but also require a large amount of reagents and consumables, resulting in low purification efficiency and high cost of active peptides. This series of problems makes the collection of active peptides extremely difficult, which seriously restricts the application of Dunaliella salina in the large-scale production of active peptides.

[0005] Therefore, it is of great significance to construct a Dunaliella salina transgenic system that can stably express exogenous genes, efficiently guide the secretion of active peptides, conveniently separate and purify active peptides, and screen high-efficiency Dunaliella salina, for promoting the development of Dunaliella salina in the field of bioactive substance production. SUMMARY

[0006] The application aims to provide a homologous recombination expression vector for expressing Pt5-1c active peptide in Dunaliella salina, and the expression vector can efficiently express exogenous genes in Dunaliella salina, thereby providing technical support for large-scale recombinant expression of active peptides and other bioactive substances in Dunaliella salina.

[0007] The application first provides a construction assembly for preparing an expression vector, and the recombinant expression vector prepared by using the construction assembly can efficiently express exogenous genes in Dunaliella salina, and the construction assembly comprises a CaMV 35S promoter with a nucleotide sequence of SEQ ID NO: 1. TGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATAACATGGTGGAGCACGACACTCTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACACGCTGA(SEQ ID NO: 1) Further, the construction component further comprises, from 5' end to 3' end after the promoter, an exogenous gene insertion site element, a P2A element, a CmR element and a terminator element; As an embodiment, the exogenous gene insertion site element is located at 739th to 1589th position, which can be inserted with an exogenous gene by double enzyme digestion of NcoI and PspXI; The P2A element has the following nucleotide sequence: GCCACCAACTTCAGCCTGCTCAAGCAGGCCGGCGACGTGGAGGAGAACCCGGGCCCC (SEQ ID NO: 2); The CmR element has the following nucleotide sequence: ATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAGTTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACAATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTTTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAA (SEQ ID NO: 3); The terminator has the nucleotide sequence as follows: GATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATC (SEQ ID NO: 4); A specific nucleotide sequence of the construction component is as follows: One of the inserted foreign genes is GFP, and the specific nucleotide sequence of its construction component is as follows: The nucleic acid sequences of the primers used to amplify the fragments when constructing the assembly are as follows: GFP-for: 5'-tgtcgatcgaccatgATGGTGAGCAAGGGCGAG-3' (SEQ ID NO: 7), GFP-rev: 5'-TTGGTGGCCTTGTACAGCTCGTCCATGCC-3' (SEQ ID NO: 8); P2A-for: 5'-GCCACCAACTTCAGCCTGCTCAAGCAGGCCGGCGACGTGGAGGAGAACCCGGGCCCC-3' (SEQ ID NO: 9) P2A-rev: 5'-GGGGCCCGGGTTCTCCTCCACGTCGCCGGCCTGCTTGAGCAGGCTGAAGTTGGTGGC-3' (SEQ ID NO: 10) CmR-for: 5'-GGGCCCCatggagaaaaaaatcactggatataccacc-3' (SEQ ID NO: 11) CmR-rev: 5'-GAACGATCttacgccccgccctgc-3' (SEQ ID NO: 12) NOS-for: 5'-GGGCCCCatggagaaaaaaatcactggatataccacc-3' (SEQ ID NO: 13) NOS-rev: 5'-GAACGATCttacgccccgccctgc-3' (SEQ ID NO: 14) The sequence of the synthetic GFP fragment with homology arms, GFP-HA, is as follows: TGTCGATCGACCATGATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGCCACCAA (SEQ ID NO: 15) The synthetic CmR fragment with homology arms, CmR-HA, has the following sequence: GGGCCCCATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAGTTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACAATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTTTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAAGATCGTTC (SEQ ID NO: 16) The sequence of the synthetic NOS fragment with homology arms, NOS-HA, is as follows: GGCGTAAGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCTCGAGTTTCTCCATA (SEQ ID NO: 17).

[0008] Another construct component is an exogenous gene site Pt5-1c active peptide (amino acid sequence is SEQ ID NO: 19, and the nucleotide sequence of the encoding gene is SEQ ID NO: 20), and the sequence information is as follows: The amino acid sequence of the Pt5-1C active peptide is as follows: SRMKKWAKIIEKWRKWHKKRWLAHHSATK (SEQ ID NO: 19); The nucleotide sequence of the coding gene is as follows: AGCCGGATGAAGAAGTGGGCGAAGATCATCGAGAAGTGGCGCAAGTGGCACAAGAAGCGCTGGCTCGCTCATCACTCGGCGACCAAG (SEQ ID NO: 20).

[0009] The nucleic acid sequence of the primers used for amplifying the fragment when constructing the assembly is as follows: Pt5-1C-for: 5'-TGTCGATCGACCATGGGCGCCATGCACTCGA-3' (SEQ ID NO: 21), Pt5-1C-rev: 5'-TTGGTGGCGAATTCTCACTTGGTCGCCGAG-3' (SEQ ID NO: 22); The sequence of the synthesized Pt5-1C fragment with homologous arms, i.e. Pt5-1C-HA, is as follows: TGTCGATCGACCATGGGCGCCATGCACTCGAAAAATTTGCTGCTCGCAGCACAGCTGCTGTTGCTACTCATTGGGACAGGCGTGTTTGCAGCGGACGACGATGATAAGAGCCGGATGAAGAAGTGGGCGAAGATCATCGAGAAGTGGCGCAAGTGGCACAAGAAGCGCTGGCTCGCTCATCACTCGGCGACCAAGTGAGAATTCGCCACCAA (SEQ ID NO: 23) The application also provides a homologous recombination expression vector, which comprises the above-mentioned assembly.

[0010] The application also provides a method for expressing an exogenous gene in Dunaliella salina, which comprises integrating the coding fragment of the exogenous gene into the genome of Dunaliella salina by using the above-mentioned homologous recombination expression vector.

[0011] The expression vector constructed in the application separates the exogenous gene from other genes through the P2A sequence, so that the exogenous gene is independently expressed, and interference between genes is avoided; and the FSRS (Ferroxidase Signal Recognition Sequence, ferroxidase signal recognition sequence) is introduced at the same time as the exogenous gene is inserted, which can guide the secretion of active peptides such as Pt5-1c to the extracellular space, avoiding the problems of impurity release and active peptide structure damage caused by broken cells to obtain active peptides. The expression vector constructed in the application introduces an enterokinase cleavage site, so that the active peptide can be conveniently cut and separated in the subsequent separation and purification process, reducing the consumption of reagents and consumables, improving the purification efficiency, and reducing the purification cost. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 A dujewski salt algae GFP expression vector map provided for the embodiment of the application.

[0013] Figure 2 A dujewski salt algae Pt5-1C expression vector map provided for the embodiment of the application. DETAILED DESCRIPTION

[0014] The specific embodiments of the application are further described below in conjunction with examples, and it should be pointed out that the specific embodiments described here are only for the purpose of illustrating and explaining the application, and are not limited to the application.

[0015] Example 1: Construction of dujewski salt algae GFP homologous recombination expression vector

[0016] This embodiment constructs a construction component inserted with an exogenous gene GFP, and the specific nucleotide sequence of the construction component is SEQ ID NO: 6.

[0017] The synthesized GFP fragment with a homologous arm, i.e. GFP-HA, has a sequence of SEQ ID NO: 15.

[0018] The synthesized CmR fragment with a homologous arm, i.e. CmR-HA, has a sequence of SEQ ID NO: 16.

[0019] The synthesized NOS fragment with a homologous arm, i.e. NOS-HA, has a sequence of SEQ ID NO: 17.

[0020] The following are specific construction steps.

[0021] 1. Construction of GFP expression vector The following primers are designed and synthesized: GFP-for: 5'-tgtcgatcgaccatgATGGTGAGCAAGGGCGAG-3' (SEQ ID NO: 7) GFP-rev: 5'-TTGGTGGCCTTGTACAGCTCGTCCATGCC-3' (SEQ ID NO: 8) PCR amplification was performed with the primers GFP-for (SEQ ID NO: 7) and GFP-rev (SEQ ID NO: 8) using the commercial vector pcDNA3.1 as a template. The reaction program was as follows: pre-denaturation at 98°C for 3 min; 98°C for 15 sec, 60°C for 15 sec, 68°C for 20 sec, for a total of 30 cycles; and extension at 68°C for 7 min. The PCR amplification product was about 740 bp, which was a green fluorescent protein gene GFP containing homologous arms at both ends, and was named GFP-HA (i.e., SEQ ID NO: 15). The fragment was purified by agarose gel electrophoresis and gel recovery (Qiagen kit) for standby use.

[0022] The following complementary single-stranded DNA was designed and synthesized: P2A-for: 5'-GCCACCAACTTCAGCCTGCTCAAGCAGGCCGGCGACGTGGAGGAGAACCCGGGCCCC-3' (SEQ ID NO: 9) P2A-rev: 5'-GGGGCCCGGGTTCTCCTCCACGTCGCCGGCCTGCTTGAGCAGGCTGAAGTTGGTGGC-3' (SEQ ID NO: 10) Double-stranded DNA was synthesized by annealing reaction. The annealing program was as follows: pre-denaturation at 95°C for 5 min; and slow cooling from 95°C to 95°C at a rate of 1°C / min. The PCR product was about 57 bp, which was a gene P2A, and was named P2A-HA (SEQ ID NO: 2). The fragment was purified by agarose gel electrophoresis and gel recovery (Qiagen kit) for standby use.

[0023] The following primers were designed and synthesized: CmR-for: 5'-GGGCCCCatggagaaaaaaatcactggatataccacc-3' (SEQ ID NO: 11) CmR-rev: 5'-GAACGATCttacgccccgccctgc-3' (SEQ ID NO: 12) The commercial carrier pcambia2200 was used as a template, and primers CmR-for (SEQ ID NO: 11) and CmR-rev (SEQ ID NO: 12) were used for PCR amplification, and the reaction program was as follows: 98°C for 3 min for pre-denaturation; 98°C for 15 sec, 60°C for 15 sec, 68°C for 20 sec, for a total of 30 cycles; 68°C for 7 min for extension. The PCR amplification product was about 675 bp, which was a chloramphenicol resistance gene CmR containing homologous arms at both ends, and was named CmR-HA (SEQ ID NO: 16). After agarose gel electrophoresis, the gel was recovered (Qiagen kit) and purified for standby use.

[0024] The following primers were designed and synthesized: NOS-for: 5'-GGGCCCCatggagaaaaaaatcactggatataccacc-3' (SEQ ID NO: 13) NOS-rev: 5'-GAACGATCttacgccccgccctgc-3' (SEQ ID NO: 14) The commercial carrier pcambia2201 was used as a template, and primers NOS-for and NOS-rev were used for PCR amplification, and the reaction program was as follows: 98°C for 3 min for pre-denaturation; 98°C for 15 sec, 60°C for 15 sec, 68°C for 20 sec, for a total of 30 cycles; 68°C for 7 min for extension. The PCR amplification product was about 275 bp, which was a NOS terminator containing homologous arms at both ends, and was named NOS-HA (SEQ ID NO: 17). After agarose gel electrophoresis, the gel was recovered (Qiagen kit) and purified for standby use.

[0025] Based on the above product, pcambia2200 was used as a starting vector to construct a vector that can be expressed in Dunaliella salina by homologous recombination.

[0026] pcambia2200 was digested by NcoI and PspXI, then connected by ABclonal 2x MultiF Seamless Assembly Mix, the total amount of linearized vector and each insert in the recombination reaction was 0.03 pmol, the molar ratio of the vector and the insert of GFP-HA (SEQ ID NO: 15), CmR-HA (SEQ ID NO: 16), NOS-HA (SEQ ID NO: 17) was 1:1, the molar ratio of the vector and the insert of P2A-HA (SEQ ID NO: 2) was 1:5, which constituted the expression system of D. salina, named 2200-GFP-CmR (SEQ ID NO: 6) (see the structure of the vector in Figure 1 ).

[0027] 2. Transformation of D. salina Centrifugal collection of 40 mL D. salina algae liquid, 1700 x g, 3 min. Washed with 2 mL 2x HEPES shock buffer for three times. In a 1.5 mL EP tube, after adding the following substances, place on ice for 20 min: ① 400 μl of algae liquid (adjust the concentration of algae cells to 10 6 / mL by adding shock buffer), ② final concentration of 10 μg / mL plasmid DNA, ③ salmon sperm DNA 20 μl.

[0028] 400 μL of algae suspension was divided into an electric shock cup, and the Eppendorf Eporator electroporator was used to perform electric shock transformation under the conditions of setting voltage 1500 V, actual working voltage 100 V, and pulse duration 654 ms.

[0029] After the algae liquid was shocked, it was placed in ice for 5 min, then centrifuged at 1700 x g for 3 min, 1 mL of medium was added to the centrifuge tube and cultured in the dark for 24 h, then transferred to 20 mL of medium (50 mL conical flask) for culture.

[0030] 3. Screening and identification of transformed D. salina The recovered D. salina cells were transferred to a selective medium to kill the untransformed cells. The selective medium was D. salina liquid medium containing 400 μg / mL chloramphenicol. After 5 days, the culture was centrifuged at 1700 x g for 3 min, and the supernatant was discarded. The collected algae were spread on solid culture plates containing 300 μg / mL chloramphenicol, and the resistant algae cells were allowed to grow and form resistant single algae colonies. After about 15 days of culture, single algae colonies were formed on the plates. The single algae colonies were picked and streaked on solid culture plates containing 300 μg / mL chloramphenicol to further purify and enhance the resistance of the resistant algae. After 20 days, the single algae colonies were picked and cultured in liquid medium for about 20 days, centrifuged at 1700 x g for 3 min, and the algae were collected, each with a wet weight of > 100 mg, and then frozen in liquid nitrogen for storage.

[0031] The total genomic DNA of the transgenic D. salina was extracted for molecular identification. First, PCR was used to identify the integration of the plasmid. The upstream primer used in the PCR was GFP-for (SEQ ID NO: 7), and the downstream primer was GFP-rev (SEQ ID NO: 8), and the product was the GFP gene. The reaction program was as described above. If the transformation is successful, this fragment can be amplified in the genome of the resistant D. salina, and there is no such fragment in the untransformed D. salina.

[0032] Then, primers were designed and synthesized upstream of the 5' end of the RB T-DNA repeat and downstream of the 3' end of the LB T-DNA repeat, and the primer sequences were as follows: con-F for: 5'-GTTTACCCGCCAATATATCCTGTCA-3' con-F rev: 5'-GTTTACACCACAATATATCCTGCCA-3' The pair of primers con-F for and con-F rev amplified a fragment of about 3330 bp in length in the homogenized transgenic D. salina genomic DNA, which included the homologous arms and the entire gene expression cassette. The total genomic DNA of the positive transgenic algae was used as a template for PCR amplification with primers con-F for and con-F rev.

[0033] The PCR reaction program was: 98°C for 3 min for pre-denaturation; 98°C for 15 sec, 60°C for 15 sec, 68°C for 2 min for 30 cycles; and 68°C for 7 min for extension.

[0034] The PCR product was electrophoresed and a band of 3330 bp was observed. Sequencing showed that the sequence was consistent with the vector sequence, indicating that the foreign gene had been inserted into the genome of D. salina and a high expression of GFP was obtained after induction.

[0035] Example 2: Construction of a homologous recombination expression vector of D. salina Pt5-1c

[0036] This example allows the preparation of a construction assembly for recombinantly expressing the active peptide of the foreign gene Pt5-1c, which has the nucleotide sequence of SEQ ID NO: 18; wherein the synthesized Pt5-1C fragment with a homologous arm, i.e. Pt5-1C-HA, has the sequence of SEQ ID NO: 23; the specific steps are as follows: 1. Construction of a Pt5-1c expression vector The following primers were designed and synthesized: Pt5-1C-for: 5'-TGTCGATCGACCATGGGCGCCATGCACTCGA-3' (SEQ ID NO: 21) Pt5-1C-rev: 5'-TTGGTGGCGAATTCTCACTTGGTCGCCGAG-3' (SEQ ID NO: 22) The commercial vector VB240716-1297azp was used as a template and subjected to PCR amplification with the primers Pt5-1C-for (SEQ ID NO: 21) and Pt5-1C-rev (SEQ ID NO: 22), and the reaction program was as follows: 98°C for 3 min for pre-denaturation; 98°C for 15 sec, 60°C for 15 sec, 68°C for 20 sec, for a total of 30 cycles; 68°C for 7 min for extension. The PCR amplification product was about 189 bp, which was a fragment containing FSRS (ferric oxidase signal recognition sequence, which can secrete pt5-1c to the extracellular), enterokinase cleavage site and antibacterial peptide Pt5-1C, and was named Pt5-1C-HA (SEQ ID NO: 23). The fragment was purified by agarose gel electrophoresis and gel recovery (Novizen kit) for standby use.

[0037] Based on the above product, pcambia2200 was used as the starting vector to construct a D. salina vector by homologous recombination method.

[0038] The pcambia2200 was digested by NcoI and PspXI, and then connected by homologous recombination using ABclonal 2x MultiF Seamless Assembly Mix. The total amount of linearized vector and each insert in the recombination reaction was 0.03 pmol, the molar ratio of the vector and the insert of CmR-HA (SEQ ID NO: 16) and NOS-HA (SEQ ID NO: 17) was 1:1, and the molar ratio of the vector and the insert of Pt5-1C-HA (SEQ ID NO: 23) and P2A-HA (SEQ ID NO: 2) was 1:5, to form a Dunaliella salina expression system, named 2200-Pt5-1C-CmR (SEQ ID NO: 18) (see the vector structure in Figure 2 ).

[0039] The transformation and screening process of Dunaliella salina was consistent with the above.

[0040] The total genomic DNA of the transgenic Dunaliella salina was extracted for molecular identification. First, the integration of the plasmid was identified by PCR. The upstream primer used in PCR was Pt5-1C-for (SEQ ID NO: 21), and the downstream primer was Pt5-1C-rev (SEQ ID NO: 22). The product was the FSRS, enterokinase cleavage site and the fragment of the antibacterial peptide Pt5-1C, and the reaction program was as described above. If successful transformation, this fragment can be amplified in the genome of the resistant Dunaliella salina, and there is no such fragment in the untransformed Dunaliella salina.

[0041] Then the primers were designed and synthesized upstream of the RB T-DNA repeat 5' end and downstream of the LB T-DNA repeat 3' end, and the primer sequences were as follows: con-F for: 5'-GTTTACCCGCCAATATATCCTGTCA-3' con-F rev: 5'-GTTTACACCACAATATATCCTGCCA-3' The pair of primers con-F for and con-F rev amplified the fragment of the homologous arm and the entire gene expression frame in the homogenized transgenic Dunaliella salina genomic DNA, with a length of about 2802 bp. The whole genomic DNA of the positive transgenic algae was used as a template for PCR amplification with primers con-F for and con-F rev.

[0042] PCR reaction procedure: 98℃ 3 min pre-denaturation; 98℃ 15 sec, 60℃ 15 sec, 68℃ 2 min, 30 cycles; 68℃ 7 min extension.

[0043] After electrophoresis, the PCR product showed a band of 2802 bp in size; after sequencing, the sequence was consistent with the vector sequence, It is indicated that the foreign gene Pt5-1C has been inserted into the genome of Dunaliella salina.

[0044] The recombinant expression results show that the recombinant expression vector constructed in this embodiment can efficiently express Pt5-1c polypeptide in Dunaliella salina.

Claims

1. A construction kit for the preparation of an expression vector, characterized in that The CaMV 35S promoter in the construction component has a nucleotide sequence of SEQ ID NO:

1.

2. The build assembly of claim 1, wherein, The construction component further comprises, from 5' end to 3' end, an insertion site element of an exogenous gene, a P2A element, a CmR element and a terminator element after the promoter.

3. The build assembly of claim 1, wherein, The nucleotide sequence of the construction component is SEQ ID NO:

5.

4. The build assembly of claim 1, wherein, The construction component further comprises an exogenous gene GFP, and the nucleotide sequence of the construction component comprising the exogenous gene GFP is SEQ ID NO:

6.

5. The build assembly of claim 1, wherein, The construction component further comprises an exogenous gene Pt5-1c active peptide, and the nucleotide sequence of the construction component comprising the exogenous gene Pt5-1c active peptide is SEQ ID NO:

18.

6. Use of the construction component of claim 1 in construction of a recombinant expression vector.

7. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the construction component of claim 1.

8. The recombinant expression vector of claim 7, wherein, The recombinant expression vector comprises the construction component of claim 5.

9. A genetically engineered Dunaliella salina, characterized in that, The coding gene of the Pt5-1c active peptide is integrated into the genome of the genetically engineered D. salina by the recombinant expression vector of claim 7.

10. A method for expressing a foreign gene in Dunaliella salina, the method comprising, The method is used to integrate the coding fragment of the exogenous gene into the genome of the D. salina by the recombinant expression vector of claim 7.

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