Crayfish egg cell guide peptide, fusion protein, nucleic acid molecule, recombinant expression vector, host cell, method for guiding foreign protein into crayfish egg cell and application thereof

Abstract

The present invention relates to the fields of molecular biology and genetic engineering, and relates to crayfish egg cell guide peptides, fusion proteins, nucleic acid molecules, recombinant expression vectors, host cells, methods for guiding exogenous proteins into crayfish eggs, and applications thereof. The amino acid sequence of the crayfish egg cell guide peptide is as shown in SEQ ID NO.1. The present invention uses green fluorescent protein (eGFP) as an example protein to prove that the polypeptide can successfully deliver exogenous proteins. This breakthrough can lay a technical foundation for the introduction of gene editing tools (such as the CRISPR-Cas9 system) into crayfish oocytes, and is expected to be widely used in the fields of crayfish breeding, functional gene research, and aquaculture gene improvement.

Description

Technical Field

[0001] The invention relates to the fields of molecular biology and genetic engineering, and relates to crayfish egg cell guide peptides, fusion proteins, nucleic acid molecules, recombinant expression vectors, host cells, methods for guiding foreign proteins into crayfish eggs, and applications thereof. Background Art

[0002] As an important aquaculture species in the world, crayfish (such as Procambarus clarkii) are of great value in biological research, breeding improvement and biotechnology applications. However, gene editing technology for crustaceans such as crayfish has long faced many challenges, especially in how to effectively deliver exogenous gene editing tools (such as CRISPR-Cas9 system) to oocytes or embryonic cells. This problem has greatly limited the application of gene editing technology in species such as crayfish.

[0003] Common gene editing tools, such as CRISPR-Cas9, TALENs or ZFN, rely on efficient delivery systems to ensure their effectiveness in target cells. Traditional methods are mainly based on microinjection of embryos, which rely on expensive instruments, professional personnel and mature systems, and are inefficient and species-limited. Therefore, this method is difficult to be widely used in crayfish research and production.

[0004] In insects and other arthropods, when egg cells and ovaries develop, a large amount of vitellogenin is absorbed into the egg cells through receptor-mediated endocytosis to provide nutrition for the development of the eggs. A sequence on vitellogenin that can be recognized by receptors is the key to determining this endocytosis, and this polypeptide therefore has the potential to guide other exogenous proteins into the egg cells. Based on this, in 2018, the Rasgon research team at the University of Pennsylvania developed a method called "Receptor-mediated Ovarian Transduction Cargo" (ReMOT Control), using a polypeptide called P2C to deliver CRISPR-Cas9 ribonucleoprotein (RNP) to the reproductive system of mosquitoes, successfully achieving heritable gene editing. Therefore, the development of ReMOT Control technology provides the possibility of delivering exogenous proteins to egg cells of other arthropods and even achieving gene editing. However, the polypeptides currently used in ReMOT control technology are not universal and difficult to be directly and universally applied. It is necessary to screen and optimize specific guide peptides for specific species. Therefore, in order to effectively guide exogenous proteins into crayfish egg cells, explore the application of ReMOT control gene editing technology in crayfish, and study the functions of other proteins in crayfish egg cells, it is urgent to carry out specific sequence screening and optimization to find polypeptides that can effectively guide exogenous proteins into crayfish egg cells. Summary of the invention

[0005] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide a crayfish egg cell guide peptide, a fusion protein, a nucleic acid molecule, a recombinant expression vector, a host cell, a method for guiding exogenous proteins into crayfish eggs, and their applications. The present invention uses green fluorescent protein (eGFP) as an example protein to prove that the polypeptide can successfully deliver exogenous proteins. This breakthrough can lay a technical foundation for the introduction of gene editing tools (such as the CRISPR-Cas9 system) into crayfish oocytes, and is expected to be widely used in the fields of crayfish breeding, functional gene research, and aquaculture gene improvement.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] Firstly, the present invention provides a crayfish egg cell guiding peptide, the amino acid sequence of which is DKNVVRPSYGAYKYIEGKQESTFRGGGGSGGGGSGGGGS, as shown in SEQ ID NO.1.

[0008] Experiments have shown that the crayfish egg cell guiding peptide provided by the present invention can introduce foreign proteins into crayfish eggs, retain the functions of the foreign proteins, and enable the foreign proteins to play corresponding roles in the crayfish eggs.

[0009] Secondly, the present invention also provides a fusion protein, which comprises the above-mentioned crayfish egg cell guiding peptide and foreign protein in sequence from N-terminus to C-terminus.

[0010] In some embodiments, the exogenous protein is green fluorescent protein (eGFP).

[0011] The present invention also provides a nucleic acid molecule, which is used to encode the above-mentioned fusion protein of the present invention.

[0012] The coding nucleic acid sequence of the nucleic acid molecule of the present invention is codon optimized. The present invention optimizes the codons of the nucleic acid molecule according to the engineered bacteria to be expressed.

[0013] In some embodiments, the nucleic acid sequence in the nucleic acid molecule for encoding the crayfish egg cell signal peptide is: gataaaaatgttgttcgtccgagctatggtgcctataaatatattgaaggtaaacaggaaagcaccttccgtggtggtggcggcagcggtggtggcggcagcggtggtggcggcagc, as shown in SEQ ID NO.2.

[0014] The present invention also provides a recombinant expression vector, which contains the nucleic acid molecule of the present invention.

[0015] The recombinant expression vector of the present invention is obtained by effectively connecting the above-mentioned nucleic acid molecule to an expression vector, and the expression vector is any one or more of a plasmid, a phage, a phagemid, a cosmid, a viral vector, an F cosmid, etc. Specifically, the recombinant expression vector can be obtained by obtaining the coding region gene fragments of the crayfish egg cell signal peptide and the foreign protein by single-segment amplification, and then connecting the expression vector in segments, or by constructing the genes encoding the above-mentioned crayfish egg cell signal peptide and the foreign protein into a fusion gene (i.e., the above-mentioned nucleic acid molecule) and then connecting the expression vector to obtain it. In some embodiments, the expression vector is a plasmid. More specifically, the plasmid includes but is not limited to pET (such as pET-28a, pET-30a or pET-32a, etc.) or pGEX (such as pGEX-4T-1, etc.). The use of the above-mentioned plasmid in the present invention is more conducive to its expression in engineering bacteria.

[0016] The present invention also provides a host cell, wherein the host cell contains the above-mentioned recombinant expression vector or nucleic acid molecule.

[0017] The host cell of the present invention can be obtained by introducing a recombinant expression vector into the host cell by chemical transformation or electroporation.

[0018] The host cell described in the present invention can be a prokaryotic cell or a eukaryotic cell, specifically, it can be any one or more of a bacterial cell, a fungal cell or a plant cell, more specifically, including but not limited to Escherichia coli, Bacillus subtilis, lactic acid bacteria, Saccharomyces cerevisiae or Streptomyces, etc.

[0019] In order to obtain the target host cells, it is necessary to screen positive clones for verification. The screening can be based on antibiotic screening, blue-white screening, PCR screening, enzyme screening or sequencing screening, etc., which are not limited here and can be selected according to actual conditions.

[0020] The purpose of the above-mentioned nucleic acid molecules, recombinant expression vectors or host cells provided by the present invention is to express the above-mentioned fusion protein. When inducing the host cell to express the fusion protein, it can be based on IPTG induction, pH / temperature induction, ester compound induction or metal ion induction, etc., which are not limited here, and can be flexibly selected according to the characteristics of the expression vector and the engineered bacteria. After the host cell expresses the fusion protein, it can be obtained by ultrasonically crushing the host cell and affinity chromatography. Specifically, the affinity chromatography can be nickel affinity chromatography, glutathione affinity chromatography or metal chelate chromatography, etc., which are not limited here, and can be selected according to the characteristics of the protein.

[0021] The present invention also provides a method for guiding foreign proteins into crayfish eggs, providing the above-mentioned crayfish egg cell guiding peptide, recombining the crayfish egg cell guiding peptide with foreign proteins into recombinant proteins, or providing the above-mentioned fusion protein; and injecting the recombinant protein or fusion protein into crayfish hemolymph.

[0022] The injection of the present invention can be carried out by a micro-syringe. In some embodiments, the injection site includes but is not limited to the ventral side of the tail, the base of the walking legs, the dorsal side or the ventral side of the head and thorax.

[0023] In some embodiments, the crayfish is a female shrimp in the gonadal maturity stage.

[0024] In some embodiments, the crayfish is Procambarus clarkii (crayfish).

[0025] The present invention also provides an application of the above-mentioned crayfish egg cell guide peptide, fusion protein, nucleic acid molecule, recombinant expression vector, host cell or method in gene editing of crayfish. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0027] Figure 1 It is a purification diagram of the eGFP protein in the embodiment of the present invention; wherein, lane M is the protein Marker, lane 1 is the protein expressed without induction, lane 2 is the protein expressed after induction with 0.2 mM IPTG, and lane 3 is the purified eGFP protein.

[0028] Figure 2 It is a purification diagram of GP-eGFP protein in an embodiment of the present invention; wherein, lane M is a protein marker, lane 1 is a protein expressed without induction, lane 2 is a protein expressed after induction with 0.2 mM IPTG, and lane 3 is a purified GP-eGFP protein.

[0029] Figure 3 These are fluorescent photos of the eGFP protein and GP-eGFP protein in the examples of the present invention.

[0030] Figure 4 This is a fluorescent picture of the crayfish ovary after the eGFP protein and GP-eGFP protein were injected into the crayfish ovary in an example of the present invention. DETAILED DESCRIPTION

[0031] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0032] Example 1 Obtaining exogenous protein (eGFP) and fusion protein (GP-eGFP)

[0033] The gene fragments of crayfish egg cell guide peptide and foreign protein were obtained by PCR amplification. Specific primers were designed as shown in Table 1 below.

[0034] Table 1 Primer sequences

[0035]

[0036] The crayfish egg cell guide peptide and the foreign protein gene are connected to each other to construct a fusion gene encoding the crayfish egg cell guide peptide-foreign protein fusion protein. The amplified fusion gene and the foreign protein gene fragment are inserted into the expression vector pET-30a by homologous recombination and transformed into Escherichia coli DH5α (competent) cells. After that, the positive clones after transformation are selected and verified by colony PCR. The recombinant plasmid is extracted from DH5α and then transformed into competent Rosetta (competent) cells. After that, the positive clones after transformation are selected and verified by colony PCR and sequencing to ensure the successful insertion of the target gene.

[0037] Among them, the amino acid sequence of the crayfish egg cell guiding peptide is: DKNVVRPSYGAYKYIEGKQESTFRGGGGSGGGGSGGGGS, as shown in SEQ ID NO.1.

[0038] The nucleic acid sequence of the crayfish egg cell guiding peptide is: gataaaaatgttgttcgtccgagctatggtgcctataaatatattgaaggtaaacaggaaagcaccttccgtggtggtggcggcagcggtggtggcggcagcggtggtggcggcagc, as shown in SEQ ID NO.2.

[0039] The amino acid sequence of eGFP is: MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKGDGNYKTRAEVKF EGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELY, as in SEQ Shown as ID NO.3.

[0040] The nucleic acid sequence of eGFP is:

[0041] , as shown in SEQ ID NO.4.

[0042] Then, positive clones were selected and inoculated into liquid culture medium and cultured until the logarithmic growth phase (OD 600 =0.5~0.8), and then add isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.2 mM to induce expression at 28°C for 6 hours. The cells were disrupted by ultrasound, the supernatant was collected by centrifugation and purified by His-tag protein affinity chromatography (reduction-resistant chelation type) to obtain the exogenous protein (eGFP, Figure 1 ) and fusion proteins (GP-eGFP, Figure 2 ).

[0043] Example 2 Observation of Autofluorescence of Exogenous and Fusion Proteins

[0044] Based on the characteristics (autonomous green fluorescence) of the exogenous protein (green fluorescent protein) involved in Example 1, its effectiveness was evaluated by autofluorescence observation. It was found that both the exogenous protein and the fusion protein could be observed to have obvious autofluorescence, and the fluorescence intensity of the two was similar, such as Figure 3 As shown, it also shows that the polypeptide and the linker region protein have no significant effect on certain properties of exogenous proteins (in this example, the autofluorescence properties of eGFP).

[0045] Example 3 Observation of the distribution of fluorescent signals in crayfish ovaries after injection of exogenous or fusion proteins

[0046] 100 μL and 1.5 μg / μL of the above exogenous proteins and fusion proteins were injected near the ovaries of crayfish, and the color of the ovaries was observed under a fluorescence microscope 48 hours later to preliminarily verify whether the two proteins could enter the ovaries. It was found that the ovaries of crayfish injected with eGFP had only weak fluorescence signals, while obvious fluorescence signals were observed in the GP-eGFP injected group, such as Figure 4 As shown. It is confirmed that this polypeptide has the function of efficiently guiding foreign proteins (eGFP) into oocytes. Although its effectiveness on other foreign proteins (such as CRISPR-Cas9) still needs to be explored in the future, this example provides an important proof of the function of this polypeptide. The discovery and subsequent application of this polypeptide provide the possibility of introducing Cas9 to achieve gene editing in crayfish.

[0047] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A crayfish egg cell guiding peptide, characterized in that: The amino acid sequence is shown in SEQ ID NO.

1.

2. A fusion protein, characterized in that: The invention comprises the crayfish egg cell guiding peptide and the foreign protein in sequence from the N-terminus to the C-terminus.

3. The fusion protein according to claim 2, characterized in that: The exogenous protein is green fluorescent protein.

4. A nucleic acid molecule, characterized in that: The nucleic acid molecule is used to encode the fusion protein according to claim 2 or 3.

5. The nucleic acid molecule according to claim 4, characterized in that The nucleic acid sequence encoding the crayfish egg cell signal peptide in the nucleic acid molecule is shown as SEQ ID NO.

2.

6. A recombinant expression vector, characterized in that: The recombinant expression vector contains the nucleic acid molecule according to claim 4 or 5.

7. The recombinant expression vector according to claim 6, characterized in that: The expression vector is a plasmid.

8. A host cell, characterized in that The host cell contains the recombinant expression vector according to claim 6 or 7 or the nucleic acid molecule according to claim 4 or 5.

9. A method for introducing foreign protein into crayfish egg cells, characterized in that: Provide the crayfish egg cell guiding peptide of claim 1, recombining the crayfish egg cell guiding peptide with an exogenous protein into a recombinant protein, or provide the fusion protein of claim 2 or 3; and inject the recombinant protein or fusion protein into the crayfish hemolymph.

10. Use of the crayfish egg cell guiding peptide according to claim 1, the fusion protein according to claim 2 or 3, the nucleic acid molecule according to claim 4 or 5, the recombinant expression vector according to claim 6 or 7, the host cell according to claim 7 or 8, or the method according to claim 9 in gene editing of crayfish.

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