Purification And CDNA Cloning Of Thermostable Nuclease From Lumbricus Bimastus

Object1. Separate and purify a thermostable nuclease from Lumbricus Bimastus and study on its properties.2. Clone the cDNA of the thermostable nuclease, deduce its amino acid sequence and analyze cDNA and amino acid sequence.Menthod1. Separation and purification of a thermostable nuclease from Lumbricus BimastusTake SDS-PAGE functional electrophoresis and UV spectrophotometry as activity monitoring methods. Obtain the crude enzyme solution by thermally denature earthworm tissue homogenate. Acquire the crude enzyme by ammonium sulfate fractional precipitation and 80% acetone precipitation. Purify the crude enzyme by ion exchange, hydrophobic exchange and gel filtration chromatography.2. Characterization of the nature and enzymatic properties of thermostable nuclease from Lumbricus Bimastus and determination of the killing activity against Bacteria and virusesUse isoelectric focusing electrophoresis(IEF),matrix-assisted laser desorption time of flight mass spectrometry (MALDI-TOF-MS),N-terminal Edman technique and other technology means to research the apparent characteristics, such as: Molecular weight, isoelectric point, peptide mass fingerprinting (PMF), N-terminal amino acid sequence. Through Comparative experiment under different conditions study on enzyme characteristics, such as: reaction optimum temperature, optimum pH, hot and acid-base stability. Take several common pathogenic microorganisms (including bacteria and viruses) as the experimental object to investigate its antifungal and antiviral activity using agar plate colony counting and plaque counting assay.3. cDNA cloning of the thermostable nuclease from Lumbricus BimastusEmploy rapid amplification of cDNA ends (RACE) technology for cDNA cloning, Process is as follows: extracted total RNA, reverse transcription obtained cDNA and designed mergers primers, as the upstream primer, according to the determination of the N-terminal amino acid sequence, taked ploy (A) universal primer as the downstream primer.Amplified the cDNA of the thermostable nuclease, Recovered PCR product by Cutting Gel. Connected sequencing vector, transformed into competent cells, followed by selected positive clones have been sequenced cDNA sequences (including 3 'UTR) and deduced the amino acid sequence.4. Analysis cDNA sequence and amino acid sequence of the thermostable nuclease from Lumbricus Bimastus with bioinformatics methodsAccording to the information of structure and function, analysis cDNA sequence and amino acid sequence of the thermostable nuclease from Lumbricus Bimastus with bioinformatics methods. Include: the base composition of nucleotide sequence, codon usage and restriction site analysis; amino acid sequence composition, the hydrophobic pattern and homology comparison; estimate protein molecular weight and isoelectric point as well as secondary structure prediction and chemical modification sitesResult1. Separated and purified a thermostable nuclease from Lumbricus Bimastus, more than 95% purity, concentration is 0.28 mg/ml.2. Measured enzyme precise molecular weight is 29897.322Da and isoelectric point is 6.11. N-terminal 13 amino acids are PLGPYKPKCDEWV. According to comparison peptide mass fingerprinting (PMF) in the protein database, there is no search to match any protein, may be a new unreported protein.3. Through comparative experiment under different conditions, identified the optimal activity temperature is 25℃, the optimum pH value between pH5.0-5.5; of heat-stable, 70℃insulation 60min still have more than 85% residual activity; nuclease is stable between pH5.0-5.5 ,Mg²⁺, Mn²⁺, Ca²⁺ has activation effect on the enzyme, Zn²⁺, Fe²⁺, Cu²⁺ has inhibitory effect on the enzyme; not sensitive to acetone and ethanol of common organic solvents methanol. Linear DNA is best substrate to the thermostable nuclease from Lumbricus Bimastus; Km value is 0.048mg/ml with single-stranded linear DNA as substrate.4. The thermostable nuclease from Lumbricus Bimastus has fungicidal effect against Escherichia, Staphylococcus, Candida albicans,hepatitis b virus and influenza virus in different extent. The fungicidal effects against Candida albicans and influenza virus are relatively weak.5. Cloned the cDNA of thermostable nuclease from Lumbricus Bimastus, which containing 3- 'UTR of the 864bp cDNA sequence, contains a 729bp of the open reading frame ORF (1-729bp), encoding 243 amino acids6. With biological software analysis, obtained cDNA sequence base composition, codon usage and restriction site data, as well as detailed theoretical protein molecular weight, isoelectric point and amino acid composition. Protein stability analysis concluded that the nature of stability.Using BLAST software, compared amino acid sequences of thermostable nuclease from Lumbricus Bimastus with the sequences of protein sequence database, homology search to the very poor did not match any protein. Protein secondary structure was informed mainly by the random coil component (about 50%), 5α-helix centers (about 20%), and the remaining 20% of the lamellar structure by biological software analysis Conclusion1. Successfully purifid a nuclease from Lumbricus Bimastus, characteristics study has confirmed its high thermal stability.2. The nature and enzymatic properties of thermostable nuclease from Lumbricus Bimastus is quite different form known nuclease. It is able to kill a variety of pathogenic microorganisms with different levels.3. Successfully cloned cDNA fragment of the thermostable nuclease from Lumbricus Bimastus...