Pagrosomus major antibacterial peptide gene, and recombinant yeast expression vector and its preparing method

A yeast expression vector, recombinant yeast technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, genetic engineering, etc., can solve problems such as unreported

Inactive Publication Date: 2005-07-20
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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Problems solved by technology

However, there have been no international reports on the construction of fish antim

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  • Pagrosomus major antibacterial peptide gene, and recombinant yeast expression vector and its preparing method
  • Pagrosomus major antibacterial peptide gene, and recombinant yeast expression vector and its preparing method
  • Pagrosomus major antibacterial peptide gene, and recombinant yeast expression vector and its preparing method

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Embodiment approach

[0030] Taking the cloning of the red sea bream antimicrobial peptide hepcidin gene and the construction of the expression vector as an example, the embodiments of the present invention will be described in detail in conjunction with the accompanying drawings.

[0031] 1. Construction of cDNA library: The cDNA library was constructed at the Yellow Sea Fisheries Research Institute from April to July 2002. Total RNA was extracted from red sea bream spleen with TRIzol kit, and Oligotex TM The spin column kit was used to separate Poly-(A) RNA, and the pBluescript II XR cDNA library kit was used to construct a cDNA library containing 1.5×10 6 clone.

[0032]2. EST sequence analysis and red sea bream antimicrobial peptide gene sequence: Plasmid DNA was prepared by a rapid micropreparation method, and the plasmid preparation was completed on a 96-well plate; the sequence of the cDNA fragment was determined using a T3 sequencing kit, and T3 and T7 primers were used to obtain the seque...

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Abstract

The present invention is red sea bream antibacterial peptide gene and recombinant yeast expression vector and their preparation process. Through library screening and EST sequence analysis to clone red sea bream antibacterial peptide (hepcidin) gene, recombining the gene to yeast expression vector pPICZalphaA to constitute antibacterial peptide gene expression plasmid pPICZ-rsbHEPC1, and transforming Pichia yeast, yeast strain with stable expression of red sea bream antibacterial peptide is obtained. Bacterium growth inhibiting experiment shows that the recombinant antibacterial peptide has dose dependent inhibiting activity on colibacillus, bacillus pyocyaneus and other two kinds of bacteria. The present invention is suitable for cloning various fish antibacterial peptide gene and expression vector constitution, and the produced antibacterial peptide is used as feed additive for the disease prevention and treatment of fishes and other aquatic animal.

Description

Technical field: [0001] The invention belongs to the field of fish genetic engineering in the field of biotechnology, and relates to a fish antibacterial peptide gene and yeast recombinant expression vector and a preparation method thereof which can be used in fish disease prevention and control. Background technique: [0002] Fish is a vertebrate with both specific and nonspecific immunity. However, compared with mammals, the specific immune system of fish is not yet developed, and the specific immune mechanism is not perfect. Therefore, non-specific immunity plays an important role in the defense response of fish to external stimuli and pathogenic organisms. It is known that the factors involved in fish non-specific immune response mainly include some phagocytic cells such as macrophages, granulocytes and cytotoxic cells, as well as protein molecules that mediate a series of immune or stress responses, such as complement, cell Factors, chemokines, lysozyme and antimicrob...

Claims

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Application Information

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IPC IPC(8): C07K14/46C12N15/09C12N15/10C12N15/12C12N15/66C12N15/81C12P21/02
Inventor 陈松林徐美瑜隋少飞
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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