C-type agglutinin gene of procambarus clarkia as well as preparation method and applications thereof

A technology of Crayfish and lectin, which is applied in the field of biological genetic engineering, can solve the problems of difficult to carry out research, difficult to raise, and urgent research.

Active Publication Date: 2012-01-18
HUBEI TAIYANGHONG BIOLOGICAL TECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In recent years, due to the epidemic infection of WSSV, the aquaculture industry has been severely damaged and suffered huge losses, which makes the research on WSSV urgent. However, there is no shrimp cell line, and shrimp is expensive, difficult to raise, and related research is difficult to carry out

Method used

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  • C-type agglutinin gene of procambarus clarkia as well as preparation method and applications thereof
  • C-type agglutinin gene of procambarus clarkia as well as preparation method and applications thereof
  • C-type agglutinin gene of procambarus clarkia as well as preparation method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0103] A method for preparing a C-type lectin gene of Procambarus clarkii, the steps of which are:

[0104] Extraction of total RNA: Take 100ug of Procambarus clarkii hepatopancreas, add 1ml TRIzol Reagent RNA extraction reagent (Invitrogen), grind thoroughly with a grinding rod, and perform other specific operations according to the instructions of Invitrogen’s TRIzol Reagent RNA extraction reagent.

[0105] Synthesis of the first strand of cDNA: Take 5ug of total RNA in (1) above, use 1ul Oligo(dT)20 as primer, 2uldNTP (10mM), 4ul 5×RT buffer, 1ul Rnase Inhibitor, 1ul ReverTra Ace reverse transcriptase , add ddH2O to a final volume of 20ul (according to TOYOBO's reverse transcription kit (ReverTra Ace-α-cDNA kit) instructions), the reverse transcription program is (42 ° C 60 min, 99 ° C 5 min, 4 ° C 5 min), cDNA was obtained.

[0106] Acquisition of partial nucleotide sequence of C-type lectin gene (Pc-Clec): design a pair of primers ClecF and ClecR according to the literatur...

Embodiment 2

[0129] A prokaryotic expression strain Escherichia coli BL21(DE3)pET-28a-mClec capable of expressing the C-type lectin of Procambarus clarkii was obtained. The whole construction process is shown in Fig. 1, and the steps are as follows:

[0130] Primer design:

[0131] Using the biological software SignalP (SignalP 3.0) to analyze the obtained protein sequence encoded by the complete open reading frame ORF of the C-type lectin gene (Pc-Clec), it was found that the C-type lectin (Pc-Clec) of Procambarus clarkii ) has a 17-amino acid signal peptide at its N-terminus, so according to the sequence of the C-type lectin gene (Pc-Clec) of Procambarus clarkii, primers were designed to amplify its mature peptide nucleotide code sequence:

[0132] mClecF: 5'-ATAGCTAGCGATTCTCCAATAAACAAGGCCAGAT-3'(Nhe I)

[0133] mClecR: 5'-GCTGGATCCTAAAGTACTGTGTATTCACAAATGG-3'(BamH I)

[0134] The present invention chooses to insert the lectin gene between the Nhe I and BamH I sites of the pET28a(+) v...

Embodiment 3

[0139] The expression and purification of the recombinant Procambarus clarkii C-type lectin protein mClec, the steps are:

[0140] Induced expression and SDS-PAGE identification of recombinant C-type lectin protein (mClec):

[0141] Inoculate the correctly identified pET28a-mClec / BL21(DE3) into a Erlenmeyer flask containing 15ug / ml kanamycin in 20ml LB medium, and culture overnight at 37°C with shaking. On the next day, take 1ml of the culture and inoculate it into a Erlenmeyer flask containing 20ml of LB medium with the same resistance, culture with shaking at 37°C until OD600=0.6, add IPTG at a final concentration of 1.0mmol / L, induce at 37°C for 4h, and centrifuge at 12 000g , 4°C, 10min to collect the bacteria, resuspended with 10ml lysis Buffer (50mM Tris-HCl, 5mM EDTA, 150mM NaCl, PH8.0) and sonicated, centrifuged to collect the supernatant and precipitate, and detected the expression of the target protein by SDS-PAGE electrophoresis. SDA-PAGE results such as Figure 4...

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Abstract

The invention discloses a C-type agglutinin gene of procambarus clarkia as well as a preparation method and applications thereof. The method comprises the following steps: A. extracting total RNA (ribose nucleic acid); B. synthetizing a first chain cDNA; C. acquiring partial nucleotide sequences; D. acquiring the nucleotide sequences at 3'-RACE: 3' end; E. acquiring the nucleotide sequences at 5'-RACE: 5' end; F. acquiring the complete nucleotide sequences of the gene; and G. coding an amino acid sequence of protein by the C-type agglutinin gene. From the cloning in procambarus clarkia to theC-type agglutinin gene, the gene is cloned to a PET-28a carrier, recombinant protein mClec is expressed in colon bacillus BL21 (DE3), and the purified mClec expresses agglutination effect to multiplebacteria and rabbit red blood cells; and the protein mClec or recombinant agglutinin obtained from cloning prokaryotic expression vector on the agglutinin gene can be used for producing shrimp and fish antivirus and antibacterial medicaments.

Description

technical field [0001] The invention relates to the technical field of biogenetic engineering, more specifically to a C-type lectin gene of Procambarus clarkii, and also to a preparation method of a C-type lectin base of Procambarus clarkii, and also to a Procambarus clarkii C-type lectin gene. Crayfish C-type lectin gene (Pc-Clec) and the use of the encoded C-type lectin polypeptide. The preparation method of the C-type lectin polypeptide specifically involves cloning the C-type lectin gene into a prokaryotic expression vector pET8a and transforming it into a prokaryotic expression host BL21(DE3), and obtaining a recombinant C-type lectin with agglutination function by inducing expression and purifying Lectin polypeptides. The invention also relates to the agglutination effect of the recombinant C-type lectin polypeptide on various bacteria and rabbit erythrocytes. Background technique [0002] Invertebrates lack an acquired immune system and rely entirely on the innate i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/10C12N1/21C12N15/63C07K14/435A23K1/16A61K38/17A61K48/00A61P31/04A61P31/10A61P31/12A61P37/02C12R1/19A23K20/153A23K50/80
Inventor 孟小林徐进平王健张清涛
Owner HUBEI TAIYANGHONG BIOLOGICAL TECH CO LTD
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