Recombinant plasmid co-expressing human antibacterial peptide and human interferon and application of recombinant plasmid in aspect of improving disease resistance of aquarium fish

Abstract

The invention belongs to the technical field of animal genetic engineering and biological breeding, and particularly relates to a recombinant plasmid for co-expressing human antibacterial peptide and human interferon and application of the recombinant plasmid in the aspect of improving the disease resistance of aquarium fish. The recombinant plasmid is a plasmid which contains a human antibacterial peptide gene hLL-37 and a human interferon gene hIFNA1 and has a Tol2 transposon, a goldfish pan-expression promoter gActb1, a green fluorescent protein gene EGFP and a polyA sequence, and the nucleotide sequence of the recombinant plasmid is as shown in SEQ ID NO. 1. The recombinant plasmid and the Tol2 transposase mRNA are jointly introduced into the aquarium fish, so that the antiviral capability and the antibacterial capability of the aquarium fish can be improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of animal genetic engineering and biological breeding technology, and particularly relates to a recombinant plasmid co-expressing human interferon and human antibacterial peptide and application thereof in improving the disease resistance of ornamental fish. BACKGROUND

[0002] Ornamental fish farming is an important part of global aquatic economic, but the intensive farming mode leads to frequent infection of pathogenic microorganisms (such as bacteria and viruses), causing high mortality and economic losses. Traditional prevention and control methods rely on antibiotics or chemical drugs, but long-term use can easily lead to drug resistance, environmental pollution and drug residue problems. In recent years, genetic engineering technology has provided a new idea for fish disease-resistant breeding, but the existing technology still faces the following bottlenecks: 1) low gene delivery efficiency: conventional transgenic methods (such as microinjection) have low integration rate, and foreign genes are easily lost, making it difficult to achieve stable inheritance; 2) limited effect of single disease-resistant factor: existing researches mainly focus on overexpression of single immune factor (such as antibacterial peptide or interferon), lacking of multi-gene synergistic disease-resistant system; 3) limited selection of promoters: some promoters have strong tissue specificity and insufficient expression intensity, making it difficult to efficiently express disease-resistant proteins in the whole fish body; 4) lack of model verification: most of the disease-resistant genetic engineering researches only aim at single pathogen infection, lacking of systematic evaluation of immune pathways (such as interferon signal) and inflammatory response. In the existing patents and literatures, transgenic technology based on antibacterial peptides such as piscidin or fish interferon can improve the disease resistance of fish, but has the problems of strong species specificity and low cross-species expression efficiency. In addition, the researches using poly (I:C) to simulate viral infection are mainly used in mammals, but the exploration of antiviral gene regulation mechanism in ornamental fish is still not deep, and there is a lack of integrated solution for synergistic defense against bacterial infection. SUMMARY

[0003] In view of the above problems, the present application aims to provide a recombinant plasmid co-expressing human antibacterial peptide and human interferon and application thereof in improving the disease resistance of ornamental fish.

[0004] The technical solution adopted by the present application is as follows: A recombinant plasmid co-expressing human antibacterial peptide and human interferon, wherein the recombinant plasmid is a plasmid with Tol2 transposon, goldfish ubiquitous promoter gActb1, green fluorescent protein gene EGFP and polyA sequence, and human antibacterial peptide gene hLL-37 and human interferon gene hIFNA1.

[0005] Further, the construction method of the above-mentioned recombinant plasmid comprises the following steps: S1: PCR-amplifying a hLL-37 gene fragment from a pcDNA3.1(+) plasmid containing the hLL-37 gene, and adding a goldfish signal peptide sequence at the 5' end of the hLL-37 gene fragment and a 6xHis tag sequence at the 3' end to obtain an optimized hLL-37 gene fragment; and PCR-amplifying a hIFNA1 gene fragment from HEK293 cells; S2: adding a P2A sequence at the 5' end of the optimized hLL-37 gene fragment to obtain a P2A-hLL-37 gene fragment; and adding a P2A sequence at the 5' end of the hIFNA1 gene fragment to obtain a P2A-hIFNA1 gene fragment; S3: connecting the P2A-hLL-37 gene fragment to a backbone plasmid with a Tol2 transposon, a goldfish ubiquitous expression promoter gActb1, a green fluorescent protein gene EGFP and a polyA sequence by seamless cloning to obtain a recombinant plasmid Tol2-gActb1-EGFP-P2A-hLL37-polyA; S4: connecting the P2A-hIFNA1 gene fragment to the Tol2-gActb1-EGFP-P2A-hLL37-polyA plasmid by seamless cloning to obtain a recombinant plasmid Tol2-gActb1-EGFP-P2A-hLL-37-P2A-hIFNA1-polyA, i.e., a recombinant plasmid co-expressing human antibacterial peptides and human interferons.

[0006] Further, the nucleotide sequence of the above-mentioned recombinant plasmid is shown in SEQ ID NO. 1.

[0007] The above-mentioned recombinant plasmid is used in the preparation of a drug for improving the disease resistance of ornamental fish.

[0008] Further, the above-mentioned ornamental fish includes zebrafish.

[0009] Further, the above-mentioned disease resistance includes antiviral ability and antibacterial ability.

[0010] The above-mentioned recombinant plasmid is used in the transgenic breeding of ornamental fish.

[0011] Further, the above-mentioned ornamental fish includes zebrafish.

[0012] A transgenic breeding method of ornamental fish includes the following steps: S1: introducing the recombinant plasmid of any one of claims 1-3 and Tol2 transposase mRNA into wild-type ornamental fish by microinjection, and screening out individuals emitting green fluorescence under a fluorescence microscope to obtain F0 generation positive individuals; S2: Cross the F0 generation positive individuals with wild type ornamental fish, and screen out the individuals emitting green fluorescence under a fluorescence microscope to obtain F1 generation positive individuals, that is, hLL-37 and hIFNA1 double transgenic ornamental fish.

[0013] The transgenic breeding method of ornamental fish according to claim 9, wherein the ornamental fish comprises zebrafish.

[0014] The application provides a double-gene co-expression system of human antibacterial peptide gene hLL-37 and human interferon gene hIFNA1 mediated by Tol2 transposon, and the disease resistance of ornamental fish is improved through the following technical breakthroughs: 1) Efficient integration and stable expression: the Tol2 transposon is combined with the goldfish ubiquitous promoter gActb1 to ensure efficient and stable expression of the exogenous gene in the whole fish body; 2) Cross-species synergistic disease resistance: the human interferon hIFNA1 can activate the conserved JAK-STAT antiviral pathway of fish, and the human antibacterial peptide hLL-37 can enhance the broad-spectrum antibacterial ability by destroying the membrane structure of pathogens, and the two can form a double barrier of "antiviral-antibacterial"; 3) Multi-dimensional verification model: the advantages of transgenic fish in inflammation regulation, immune activation and survival rate are systematically evaluated through poly (I:C) induction to simulate viral infection and E. coli challenge experiment. The application provides an efficient and environmentally friendly genetic engineering solution for disease resistance breeding of ornamental fish, significantly reduces the dependence on chemical drugs, and lays a foundation for analyzing the functional mechanism of cross-species immune factors in fish. The technical framework can be extended to the disease resistance improvement of other economic fish, and has important scientific value and industrial application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 : Technical flow chart for integrating human antibacterial peptide gene hLL-37 and human interferon gene hIFNA1 to improve the disease resistance of ornamental fish.

[0016] Figure 2 : Structure diagram of Tol2-gActb1-EGFP-P2A-hLL-37-P2A-hIFNA1-polyA plasmid.

[0017] Figure 3 : Phenotypic trait analysis results of wild type zebrafish and hLL-37 and hIFNA1 double transgenic zebrafish.

[0018] Figure 4 : Statistical results of yolk sac area, heart rate, body length and pericardial area of wild type zebrafish and hLL-37 and hIFNA1 double transgenic zebrafish.

[0019] Figure 5 PCR amplification results of hLL-37 gene fragment (A) and hIFNA1 gene fragment (B) in wild type zebrafish, hLL-37 and hIFNA1 double transgenic zebrafish.

[0020] Figure 6 mRNA expression levels of immune-related genes in wild type zebrafish, hLL-37 and hIFNA1 double transgenic zebrafish after poly (I:C) induction.

[0021] Figure 7 Survival rates and mRNA expression levels of immune-related genes of wild type zebrafish, hLL-37 and hIFNA1 double transgenic zebrafish after E. coli infection. DETAILED DESCRIPTION

[0022] In order to make the content of the present application more convenient to understand, the technical solutions of the present application will be further described below in combination with specific embodiments, but the present application is not limited to this.

[0023] The experimental methods used in the following examples are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0024] It should be noted that the recombinant plasmid obtained by the inventors of the present application during the research and development process is obtained by adjusting the sequence of genes, fragments and various cross combinations. The skilled person can also directly synthesize according to the disclosure of the present application, which does not affect the implementation of the present application, but the cost will be increased.

[0025] The primers used by the inventors of the present application during the research and development process are as follows: LL-37-F: 5'-GAGCTGAGAGTGGCAAGCAT-3' Tol2-F: 5'-CCCCTGAACCTGAAACATAAAATG-3' Tol2-R: 5'-CTTGTACAGCTCGTCCATGCCG-3' EG-P2A-TY-F: 5'-GACGAGCTGTACAAGGGATCCGGAGCTACTAATTT-3' LL-37-Tol2-TY-R: 5'-TTTCAGGTTCAGGGGATGATGATGATGATGATGGC-3' TOL2-B-EG-LL-37-GJ-R: ATGATGATGATGATGATGGC-3' LL-37-P2A-F: 5'-GCCATCATCATCATCATCATGGATCCGGAGCTACT-3' Tol2-IFNA1-R: 5'-AGGTTCAGGGGTTATTCCTTCCTCCTTAATCTTTC-3' Example 1 S1: Cloning of human antibacterial peptide gene hLL-37 and human interferon gene hIFNA1 The pcDNA3.1(+) plasmid containing the hLL-37 gene was used as a template to amplify the hLL-37 gene fragment by PCR, and a goldfish signal peptide sequence was added to the 5' end of the hLL-37 gene fragment, and a 6xHis tag sequence was added to the 3' end of the hLL-37 gene fragment to obtain an optimized hLL-37 gene fragment. The total RNA of HEK293 cells was extracted and reverse transcribed into cDNA as a template to amplify the hIFNA1 gene fragment by PCR. The PCR amplification reaction system was as follows: 2xprimer star mix 25 μL, 10 μM of upstream primer 2.5 μL, 10 μM of downstream primer 2.5 μL, template 1 μL, H2O 19 μL; the PCR amplification reaction conditions were as follows: 95 °C for 3 min; 95 °C for 15 s, 55 °C for 15 s, 72 °C for 30 s, 35 cycles; 72 °C for 5 min; 4 °C for 10 min.

[0026] S2: Construction of hIFNA1 gene and hLL-37 gene co-expression plasmid S2-1: A P2A sequence was added to the 5' end of the optimized hLL-37 gene fragment by PCR amplification to obtain a P2A-hLL-37 gene fragment. A P2A sequence was added to the 5' end of the hIFNA1 gene fragment by PCR amplification to obtain a P2A-hIFNA1 gene fragment. The PCR amplification reaction system and the PCR amplification reaction conditions were the same as in S1.

[0027] S2-2: The backbone plasmid with a Tol2 transposon, a goldfish ubiquitous expression promoter gActb1, a green fluorescent protein gene EGFP, and a polyA sequence was amplified by PCR using primers Tol2-F and Tol2-R to obtain a linearized backbone plasmid. The P2A-hLL-37 gene fragment was amplified by PCR using primers EG-P2A-TY-F and LL-37-Tol2-TY-R to add homologous arms, and then the linearized backbone plasmid and the P2A-hLL-37 gene fragment were mixed and transformed into DH5α competent cells to obtain the P2A-hLL-37-Tol2 backbone plasmid. Seamless Cloning Kit no-seam cloning kit was used to connect with the linearized backbone plasmid, and the recombinant plasmid Tol2-gActbl-EGFP-P2A-hLL37-polyA was obtained. In the process, the PCR amplification reaction system and the PCR amplification reaction conditions were the same as S1; the backbone plasmid with the Tol2 transposon, the goldfish pan-expression promoter gActbl, the green fluorescent protein gene EGFP and the polyA sequence was modified from the pminiTol2 plasmid; the connection system was strictly configured according to the instructions of Seamless Cloning Kit; and the connection conditions were 50°C for 15 min.

[0028] S2-3: The recombinant plasmid Tol2-gActbl-EGFP-P2A-hLL37-polyA was subjected to PCR amplification using primers LL-37-F and TOL2-B-EG-LL-37-GJ-R, and the linearized Tol2-gActbl-EGFP-P2A-hLL37-polyA plasmid was obtained. The P2A-hIFNA1 gene fragment was subjected to PCR amplification using primers LL-37-P2A-F and Tol2-IFNA1-R to make it have a homologous arm, and then the linearized P2A-hIFNA1 gene fragment was obtained using the Seamless Cloning Kit. Seamless Cloning Kit no-seam cloning kit was used to connect with the linearized Tol2-gActbl-EGFP-P2A-hLL37-polyA plasmid, and the recombinant plasmid Tol2-gActbl-EGFP-P2A-hLL-37-P2A-hIFNA1-polyA was obtained, the nucleotide sequence of which is shown in SEQ ID NO. 1, and the structural schematic diagram thereof is shown in Figure 2 . In the process, the connection system and the connection conditions were the same as S2-2; in SEQ ID NO. 1, 47-124 bp is the polyA sequence, 159-357 bp is the Tol2R, 2788-2987 bp is the Tol2L, 2988-6230 bp is the goldfish pan-expression promoter gActbl, 6238-6954 bp is the green fluorescent protein gene EGFP, 6955-7206 bp is the P2A-hLL-37 gene fragment, and 7207-7839 bp is the P2A-hIFNA1 gene fragment.

[0029] Example 2: S1: In vitro transcription synthesis of Tol2 transposase mRNA The nucleotide sequence of the in vitro transcription synthesized Tol2 transposase mRNA is shown in SEQ ID NO. 2.

[0030] S2: Obtaining of positive F0 generation zebrafish ​The nucleotide sequence of the Tol2-gActbl-EGFP-P2A-hLL-37-P2A-hIFNA1-polyA plasmid with a final concentration of 50 ng / μL, as shown in SEQ ID NO. 1, was mixed with Tol2 transposase mRNA with a final concentration of 100 ng / μL, and microinjection was performed on the yolk sac of wild-type zebrafish single-cell embryos. The injected embryos were placed in culture solution and incubated at 28°C. At 120 hpf, the phenotype was observed using a fluorescence microscope, and individuals with green fluorescence signals were selected to obtain positive F0 generation zebrafish, which were then bred to sexual maturity. The positive rate was 71.5%.

[0031] S5: Obtaining of positive F1 generation zebrafish The F1 generation embryos were obtained by crossing the positive F0 generation zebrafish bred to sexual maturity with wild-type zebrafish, and were placed in culture solution and incubated at 28°C. At 120 hpf, the phenotype was observed using a fluorescence microscope, and individuals with green fluorescence signals were selected to obtain positive F1 generation zebrafish, which were hLL-37 and hIFNA1 double transgenic zebrafish.

[0032] The results of the phenotype trait analysis of wild-type zebrafish and hLL-37 and hIFNA1 double transgenic zebrafish are shown in Figure 3 Figure 3 A can be seen that, compared with wild-type zebrafish, hLL-37 and hIFNA1 double transgenic zebrafish can be seen to have systemic pan-expression of green fluorescence. From Figure 3 B-3C can be seen that the survival rate of hLL-37 and hIFNA1 double transgenic zebrafish is slightly lower than that of wild-type zebrafish, the hatching rate is slightly higher than that of wild-type zebrafish at 48 hpf but is significantly lower than that of wild-type zebrafish at 72 hpf.

[0033] The yolk sac area, heart rate, body length, and pericardial area of wild-type zebrafish and hLL-37 and hIFNA1 double transgenic zebrafish at 120 hpf were further statistically analyzed, and the results are shown in Figure 4 As can be seen from the figure, the yolk sac area, heart rate, body length, and pericardial area of hLL-37 and hIFNA1 double transgenic zebrafish had no significant difference from those of wild-type zebrafish.

[0034] Total RNA was extracted from wild-type zebrafish and hLL-37 and hIFNA1 double transgenic zebrafish at 120 hpf, and reverse transcribed into cDNA as a template for PCR amplification to detect whether they carried hLL-37 gene fragments and hIFNA1 gene fragments, and the results are shown in Figure 5 ​As shown in the figure, the hLL-37 gene fragment and the hIFNA1 gene fragment were successfully detected in the hLL-37 and hIFNA1 double transgenic zebrafish, revealing that the hLL-37 gene fragment and the hIFNA1 gene fragment in the hLL-37 and hIFNA1 double transgenic zebrafish can be co-transcribed with the green fluorescent protein gene EGFP.

[0035] To explore the effect of the hLL-37 and hIFNA1 double transgenic zebrafish in the anti-virus aspect, 1 nL of 10 mg / mL poly(I:C) was injected into the yolk sac of the 48 hpf embryos of the wild type zebrafish and the hLL-37 and hIFNA1 double transgenic zebrafish by microinjection, and the same volume of PBS was injected as a control. After injection, the embryos were placed in the culture solution and incubated at 28°C. At 120 hpf, total RNA was extracted, reverse transcribed into cDNA as a template, and the mRNA expression levels of the immune-related genes rsad2 (Gene ID: 570456), hexb (Gene ID: 323613), itk (Gene ID: 798906), cpa5 (Gene ID: 246092), isg15 (Gene ID: 558956), cnbpa (Gene ID: 326846), and cnbpb (Gene ID: 335839) were detected by RT-qPCR. The results are shown in the figure. Figure 6 As shown in the figure, the expression levels of the rsad2, itk, and isg15 genes in the poly(I:C) group of the wild type zebrafish had no significant difference with those in the PBS group of the wild type zebrafish, and the expression levels of the hexb, cnbpa, and cnbpb genes were significantly higher than those in the PBS group of the wild type zebrafish. The expression level of the rsad2 gene in the poly(I:C) group of the hLL-37 and hIFNA1 double transgenic zebrafish had no significant difference with that in the poly(I:C) group of the wild type zebrafish, the expression level of the itk gene was significantly higher than that in the poly(I:C) group of the wild type zebrafish, and the expression levels of the hexb, isg15, cnbpa, and cnbpb genes were significantly lower than those in the poly(I:C) group of the wild type zebrafish. It can be seen that the expression of the immune-related genes in the hLL-37 and hIFNA1 double transgenic zebrafish was affected after induction by poly(I:C).

[0036] To explore the effect of hLL-37 and hIFNA1 double transgenic zebrafish in antibacterial aspect, 1 nL of 100 cfu / nL of E. coli was injected into the yolk sac of wild type zebrafish, hLL-37 and hIFNA1 double transgenic zebrafish 48 hpf embryos by microinjection, and the same volume of PBS was injected as a control. The injected embryos were placed in the culture solution and incubated at 28°C. The mRNA expression levels of immune related genes were detected 1 h after injection by the method described above, and the survival rate of embryos after E. coli infection was counted, and the results are shown in Figure 7 As shown in Figure 7 A, the survival rate of hLL-37 and hIFNA1 double transgenic zebrafish in the E. coli group was significantly higher than that of wild type zebrafish in the E. coli group. As shown in Figure 7 B-7G, the expression levels of rsad2 and cnbpb genes in wild type zebrafish in the E. coli group had no significant difference with those in wild type zebrafish in the PBS group, the expression levels of itk, hexb and cpa5 genes were significantly lower than those in wild type zebrafish in the PBS group, and the expression level of cnbpa gene was significantly higher than that in wild type zebrafish in the PBS group; the expression levels of rsad2, cnbpb and cnbpa genes in hLL-37 and hIFNA1 double transgenic zebrafish in the E. coli group were significantly lower than those in wild type zebrafish in the E. coli group, the expression levels of itk, hexb, cpa5 and cnbpb genes were significantly higher than those in wild type zebrafish in the E. coli group, and the expression level of cnbpa gene had no significant difference with that in wild type zebrafish in the E. coli group. It can be seen that the expression of immune related genes in hLL-37 and hIFNA1 double transgenic zebrafish was affected after E. coli infection.

Claims

1. A recombinant plasmid co-expressing human antimicrobial peptide and human interferon, characterized in that: The recombinant plasmid contains a human antimicrobial peptide gene. hLL-37 and human interferon gene hIFNA1 With Tol2 transposon, goldfish generic expression promoter gActb1 Green fluorescent protein gene EGFP and polyA The sequence is a plasmid.

2. The recombinant plasmid according to claim 1, characterized in that: The method for constructing the recombinant plasmid includes the following steps: S1: From containing hLL-37 PCR amplification of the gene in the pcDNA3.1(+) plasmid was obtained. hLL-37 Gene fragments, and in hLL- 37 By adding a goldfish signal peptide sequence to the 5' end of the gene fragment and a 6×His tag sequence to the 3' end, an optimized sequence was obtained. hLL-37 Gene fragment; obtained by PCR amplification from HEK293 cells hIFNA1 Gene fragments; S2: In the optimized version hLL-37 Adding a P2A sequence to the 5' end of a gene fragment yields... P2A-hLL-37 Gene fragments; in hIFNA1 Adding a P2A sequence to the 5' end of a gene fragment yields... P2A-hIFNA1 Gene fragments; S3: Will P2A-hLL-37 Gene fragments were ligated to a goldfish-specific promoter containing the Tol2 transposon via seamless cloning. gActb1 Green fluorescent protein gene EGFP and polyA The backbone plasmid of the sequence was used to obtain the recombinant plasmid Tol2-gActb1-EGFP-P2A-hLL37-polyA; S4: Will P2A-hIFNA1 The gene fragment was ligated into the plasmid Tol2-gActb1-EGFP-P2A-hLL37-polyA via seamless cloning to obtain the recombinant plasmid Tol2-gActb1-EGFP-P2A-hLL-37-P2A-hIFNA1-polyA, which is a recombinant plasmid co-expressing human antimicrobial peptide and human interferon.

3. The recombinant plasmid according to claim 2, characterized in that: The nucleotide sequence of the recombinant plasmid is shown in SEQ ID NO.

1.

4. The use of the recombinant plasmid as described in any one of claims 1 to 3 in the preparation of a drug to enhance the disease resistance of ornamental fish.

5. The application according to claim 4, characterized in that: The ornamental fish include zebrafish.

6. The application according to claim 4, characterized in that: The disease resistance includes antiviral and antibacterial capabilities.

7. The application of the recombinant plasmid as described in any one of claims 1 to 3 in the transgenic breeding of ornamental fish.

8. The application according to claim 7, characterized in that: The ornamental fish include zebrafish.

9. A method for transgenic breeding of ornamental fish, characterized in that: Includes the following steps: S1: The recombinant plasmid described in any one of claims 1 to 3 and Tol2 transposase mRNA are introduced into wild-type ornamental fish by microinjection, and individuals that emit green fluorescence are screened under a fluorescence microscope to obtain F0 generation positive individuals; S2: F0 generation positive individuals are crossed with wild-type ornamental fish. Individuals that emit green fluorescence are selected under a fluorescence microscope to obtain F1 generation positive individuals, which are... hLL-37 and hIFNA1 Double genetically modified ornamental fish.

10. The method for transgenic breeding of ornamental fish according to claim 9, characterized in that: The ornamental fish include zebrafish.

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