阅读说明：本技术 海马孵化酶基因及其编码蛋白和应用 (Hippocampus japonicus hatching enzyme gene and coding protein and application thereof ) 是由 张辉贤 林强 张博 张艳红 于 2019-08-29 设计创作，主要内容包括：本发明公开了海马孵化酶基因及其重组蛋白和应用。本发明首次鉴定了线纹海马具有两种孵化酶HCE和LCE,且通过体外重组表达,获得了具有生物学活性的孵化酶蛋白,且发现孵化酶HCE和LCE具有消化溶解海马胚胎卵膜的作用,介导海马的孵化过程。本发明为大规模海马繁育技术提供了理论指导,具有潜在的重要经济价值和意义。(The invention discloses a hippocampal hatching enzyme gene, a recombinant protein and application thereof. The invention firstly identifies that the hippocampus kelloggi has two hatching enzymes HCE and LCE, obtains hatching enzyme protein with biological activity by in vitro recombination expression, and finds that the hatching enzymes HCE and LCE have the function of digesting and dissolving the embryo egg membrane of the hippocampus, and mediates the hatching process of the hippocampus. The invention provides theoretical guidance for large-scale hippocampal breeding technology and has potential important economic value and significance.)

1.A Hippocampus kelloggi hatching enzyme HCE, characterized in that its amino acid sequence is shown in SEQ ID NO. 1.

2. A gene encoding the hatching enzyme HCE of claim 1.

3. The gene of claim 2, wherein the nucleotide sequence is as shown in SEQ ID No. 3.

4. A Hippocampus kelloggi hatching enzyme LCE, characterized in that its amino acid sequence is shown in SEQ ID NO. 2.

5. A gene encoding a hatching enzyme LCE according to claim 4.

6. The gene of claim 5, wherein the nucleotide sequence is represented by SEQ ID No. 4.

7. A recombinant expression vector comprising the gene of claim 2, 3, 5 and/or 6.

8. A host cell comprising the recombinant expression vector of claim 7.

9. Use of a hippocampus kelloggi hatching enzyme HCE or a gene encoding the same as claimed in claim 1, and a hippocampus kelloggi hatching enzyme LCE or a gene encoding the same as claimed in claim 4, for the preparation of a preparation for promoting hippocampal embryo hatching.

10. A preparation for promoting hatching of hippocampus embryo, comprising Hippocampus kelloggi hatching enzyme HCE or a gene encoding the same according to claim 1, and Hippocampus kelloggi hatching enzyme LCE or a gene encoding the same according to claim 4.

Technical Field

The invention relates to the technical field of aquatic animal molecular biology, in particular to a hippocampal hatching enzyme gene and a coding protein and application thereof.

Background

The sea horse (Hippocampus) is a precious marine medicinal source fish and has high ornamental value; in recent years, wild hippocampus resources are sharply attenuated due to over-fishing, habitat damage and environmental pollution, and most of hippocampus species are on the verge of extinction. The rapid development of the Hippocampus culture industry is promoted by the aggravation of resource shortage and the flourishing of market demand, the artificial breeding technology of the Hippocampus is the most effective strategy for resource protection and sustainable utilization, and the Hippocampus kelloggi (hippopopus erectus) is the largest Hippocampus species in China at present. However, the artificial breeding technology of hippocampus has many problems, especially the basic research on the breeding biology of hippocampus is less, the research on the spawning and hatching mechanism of hippocampus is still limited, and sufficient theoretical guidance cannot be provided for the large-scale hippocampus breeding technology.

Hatching enzyme is a key enzyme participating in the hatching process of fishes, and the hatching process of the fishes mainly comprises two stages of dissolution and digestion of hatching enzyme on egg membranes and hatching of embryos from the egg membranes through muscle movement. Hatching enzymes of most fish species include two enzymes: one is high-egg membrane lyase (HCE) and the other is low-egg membrane Lyase (LCE). The action mechanism is as follows: HCE first expands the intact egg shell, and the inner layer of the expanded egg membrane becomes the substrate of LCE, which can be further digested by LCE. At present, no report on relevant researches of Hippocampus kelloggi hatching enzyme HCE and LCE genes is found.

Disclosure of Invention

The invention aims to provide a hippocampus kelloggi hatching enzyme gene, and a coding protein and application thereof, wherein recombinant expression protein generated by the hippocampus kelloggi hatching enzyme HCE and LCE gene has the function of digesting and dissolving a hippocampal embryonic egg membrane and mediates the hatching process of hippocampus.

In order to achieve the above purpose, the invention provides the following technical scheme:

the nucleotide sequence of the Hippocampus kelloggi hatching enzyme HCE gene is shown in SEQ ID No. 3. Correspondingly, the amino acid sequence of the protein coded by the HCE gene is shown as SEQ ID NO. 1.

The nucleotide sequence of the Hippocampus kelloggi hatching enzyme LCE gene is shown in SEQ ID No. 4. Correspondingly, the amino acid sequence of the protein coded by the LCE gene is shown as SEQ ID NO. 2.

A recombinant expression vector contains the Hippocampus kelloggi hatching enzyme HCE gene and/or LCE gene. Preferably, the vector is pET3c vector.

A host cell comprising the Hippocampus kelloggi hatching enzyme HCE gene and/or LCE gene or the recombinant expression vector. Preferably, the host cell is an E.coli cell.

The invention also provides the application of the hippocampus kelloggi hatching enzyme HCE or the coding gene thereof, or the hippocampus kelloggi hatching enzyme LCE or the coding gene thereof in preparing a preparation for promoting hippocampal embryo hatching.

A preparation for promoting hatching of hippocampus embryo, which contains Hippocampus kelloggi hatching enzyme HCE or its coding gene, or Hippocampus kelloggi hatching enzyme LCE or its coding gene.

Compared with the prior art, the invention has the advantages that:

the invention firstly identifies that the hippocampus kelloggi has two hatching enzymes HCE and LCE, obtains hatching enzyme protein with biological activity by in vitro recombination expression, and finds that the hatching enzymes HCE and LCE have the function of digesting and dissolving the embryo egg membrane of the hippocampus, and mediates the hatching process of the hippocampus. The invention provides theoretical guidance for large-scale hippocampal breeding technology and has potential important economic value and significance.

Drawings

FIG. 1 shows SDS-PAGE detection of the sizes of Hippocampus kelloggi hatching enzyme HCE and LCE recombinant proteins. Wherein, 1.HCE inclusion body; LCE inclusion bodies; 3. inducing HCE protein supernatant, and 4, inducing LCE protein supernatant; 5. (ii) an uninduced HCE protein; 6. (ii) an uninduced LCE protein; BL21(DE3) pLysE cell 8 protein Marker.

FIG. 2 shows specific substrate detection of Hippocampus kelloggi hatching enzymes HCE and LCE (MCA polypeptide substrate assay). Wherein the ordinate represents the activity index of the enzyme in nM _ AMC// CA/30min, and the abscissa represents the substrate species, 1. Boc-Val-Pro-Arg-MCA; Suc-Leu-Leu-Val-Tyr-MCA; Z-Ala-Ala-Asn-MCA; Suc-Ile-Ile-Trp-MCA; Suc-Ala-Pro-Ala-MCA; Suc-Ala-Glu-MCA; Z-Leu-Arg-Gly-Gly-MCA; Z-Val-Lys-Met-MCA; Z-Phe-Arg-MCA; Boc-Phe-Ser-Arg-MCA; Ac-Asp-Glu-Val-Asp-MCA; Bz-Arg-MCA; Suc-Gly-Pro-Leu-Gly-Pro-MCA; Boc-Val-Leu-Lys-MCA; sue (OMe) -Ala-Ala-Pro-Val-MCA; Z-Leu-Leu-Glu-MCA; Z-Leu-Leu-Leu-MCA.

FIG. 3 shows the measurement of the activity of Hippocampus kelloggi hatching enzyme HCE and LCE in digesting fertilized egg membrane. Wherein, control: hippocampal membrane without any treatment (negative control); HCE: hippocampal membrane after HCE digestion; LCE: hippocampal oocyst membrane after LCE digestion; HCE + LCE: hippocampal membrane after co-digestion with HCE and LCE.

Detailed Description

The following examples are further illustrative of the present invention and are not intended to be limiting thereof.