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Branchiostoma belcheri chitin-binding associated serine protease CASP gene for identifying chitin and application thereof

A technology of serine protease and amphioxus, applied in CASP protein and drug application field, can solve problems such as complement pathway damage, lack of hemolytic activity, suppurative infection, etc.

Inactive Publication Date: 2014-01-15
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

MASP2 deficiency can lead to loss of hemolytic activity of the lectin pathway, leading to suppurative infection; inflammatory lung disease can also lead to impairment of MASP-related complement pathways

Method used

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  • Branchiostoma belcheri chitin-binding associated serine protease CASP gene for identifying chitin and application thereof
  • Branchiostoma belcheri chitin-binding associated serine protease CASP gene for identifying chitin and application thereof
  • Branchiostoma belcheri chitin-binding associated serine protease CASP gene for identifying chitin and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Extraction of amphioxus total RNA and amplification of CASP partial fragments.

[0053] Extraction of total RNA and synthesis of RACE cDNA: Whole amphioxus was taken, total RNA was extracted with Trizol reagent, and protein was extracted by phenol / chloroform extraction to obtain total RNA of amphioxus. Take 1 μg of total RNA, and perform reverse transcription to synthesize the first strand according to the instructions of TOYOBO's First Strand cDNA Synthesis Kit ReverTra Ace-α-TM (code No. FSK-100). Take 2 μl of the first-strand cDNA product, and use the combined primers 5′PCR Primer: 5′-ACCCATCCCTCCCAGTCAC-3′ and 3′PCR Primer: 5′-GTAGACACTCGGCTTGGCG-3′ designed according to the predicted sequence of the CASP gene in the Florida amphioxus sequencing database as primers, RT-PCR was carried out to amplify the 899bp partial fragment of CASP. The amplified target fragment was connected to pGEX T easy vector (promega) and transformed into DH5α Escherichia coli, a...

Embodiment 2

[0054] Example 2: RACE amplifies the 5' and 3' ends of the CASP gene.

[0055] According to Invitrogen's GeneRacerTM Kit, perform RNA dephosphorylation RACE, decapping reaction, RNA oligo ligation and mRNA reverse transcription to synthesize a cDNA strand, and use Takara's LA Taq polymerase to perform PCR amplification according to the reaction system of GeneRacerTM Kit . Among them, according to the sequence of the CASP partial fragment amplified above, the gene-specific outer primer for 5′ RACE amplification is designed as 5′-gene-specific primer: 5′-GACCATCCAAGGCATCACCAGTT-3′; the inner nest primer is 5′ -nested primer: 5′-GGACACTGACATGGACTGAAGGAGTA-3′; CASP’s 3′ RACE amplified gene-specific coat primer is 5′-gene-specific primer: 5′-GGAAGGAGACCACCCACGGCT-3′; inner nest primer is 5′-nested primer: 5'-CGCTACGTAACGGCATGACAGTG-3'. The amplified target fragment was connected to pGEX T easy vector (promega), transformed into DH5α Escherichia coli, and the recombinant clone was...

Embodiment 3

[0056] Example 3: Amplification and sequence analysis of the full length of CASP.

[0057] According to the sequence of the CASP gene spliced ​​in Example 2, primers 5'-TCTGCATTTCATCATCAAACG-3' and 5'-TTGGGTGAGGTTTCTAAGGCTAA-3' were designed, and the whole gene of CASP was amplified by Takara's LATaq polymerase, and the obtained 1976 fragment was amplified. Electropherogram such as figure 2 shown. The amplified target fragment was connected to pGEX T easy vector (promega) and then transformed into DH5α Escherichia coli, and the recombinant clone was selected for sequencing. Blast homology analysis showed that the EST sequence encoding the whole gene of CASP was obtained.

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Abstract

The invention relates to a branchiostoma belcheri chitin-binding associated serine protease CASP gene for identifying chitin, a protein coded by the gene and an expression method and application of the protein. The CASP gene is aN MASP (Mbl Associated Serine Protease) similar gene obtained by cloning from a total RNA (Ribose Nucleic Acid) of branchiostoma belcheri by a method of combining primer amplification and RACE (rapid-amplification of cDNA ends) amplification. The protein coded by the CASP gene is expressed in an intracellular soluble form in escherichia coli by a recombinant expression vector PET-32a(+)-CASP. The protein coded by the CASP gene takes an important effect of resisting to foreign pathogen and has the development value of becoming a high-efficiency natural antibacterial medicament.

Description

technical field [0001] The invention relates to a unique MASP-like gene (named CASP gene) and its coded CASP protein, and the application of the gene and protein in the preparation of medicines for treating infectious diseases, belonging to the field of genetic engineering. Background technique [0002] The lectin pathway activated by complement is an important defense measure of innate immunity and plays important biological functions in organisms, such as cell lysis, bacteriolysis, opsonization and mediating inflammatory response. [0003] The complexes that activate the lectin pathway are determined by a series of pattern recognition molecules and related families of serine proteases. The earliest reported discovery that Mannose Bindig lectin (MBL) activates the complement pathway was more than 20 years ago. Since then, the importance of the complement system in innate immunity has been re-recognized. The current research results show that MBL-associated serine proteases...

Claims

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Application Information

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IPC IPC(8): C12N15/57C12N9/64C12N15/70A61K38/48A61P31/04
Inventor 徐安龙李锐黄盛丰赵红晨张晗黄慧清
Owner SUN YAT SEN UNIV
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