Nuclear protein is a kind of protein that is synthesized in the cytoplasm and then transported to the nucleus for action.Such as various histones, DNA synthetases, RNA transcription and processing enzymes, and various protein factors that play a regulatory role.
Nucleoproteins generally contain special amino acid signal sequences, which play a role in protein orientation and positioning.
The nucleoprotein isBinding proteinClass of.A special form of protein commonly found in the nuclei of various organisms, composed of nucleic acid andHistone、ProtamineWaitingbasic proteinCombine and become the main component of the nucleus.According to different types of nucleic acids, it can be divided intoRibonucleic acid nucleoproteinandDeoxyribonucleic acid nucleoprotein。It can be extracted from the nucleus and can be dissolved in 1mol/L NaCl solution.Proteins in nucleoproteins include histones (or protamine) andNonhistoneprotein.Histone containsBasic amino acidasArginine, lysine and other rich alkaline proteinspositive charge。Protamine only appears inEukaryotic cellMedium, containing moreAspartate、glutamateetc.Acid amino acid, Yesacidic protein , negatively charged.Nucleoprotein in genetics andprotein synthesisIt plays a decisive role inphageThe only component of.[1]
Nucleoprotein in virus
Nucleoprotein
A kind of combination of protein and nucleic acidComplex protein。It exists in all living things.Viruses are very simple creatures, and their chemical essence is nucleoprotein.Nuclear proteins in cells mainly exist inchromosomeAnd ribosome.Since nucleic acids have two types of RNA, the nucleoproteins are also divided into DNA nucleoproteins and RNA nucleoproteins.DNA nucleoprotein mainly exists in the nucleus, and RNA nucleoprotein mainly exists in the ribosome.Both types of nucleoproteins have similar physical and chemical properties and can bealkaline dyeDyeing is unstable and easy to decompose into nucleic acid and protein under the action of heat or alkali.DNA nucleoprotein has strong viscosity and is insoluble in isotonic salt solution, but soluble in salt solution with extremely low ionic strength;RNA nucleoprotein can be dissolved in isotonic salt solution.The protein component of nucleoprotein is mainly basic proteins such as histone and protamine, and other proteins such asTobacco mosaic virusThe protein molecule is very large, each molecule is composed of 2130 peptide chains, and the molecular weight of peptide chains is 17500 per day.
Ribonucleoprotein (RNP) refers to the nuclear protein containing RNA, which is a form of combining nucleic acid and protein.RibonucleoproteinIncluding ribosomestelomeraseAnd small nuclear RNP (snRNP).RNPstayinfluenza virusA plays an important role in the replication process.RiboribosomeAlso called ribosomeRiboribosome。A subcellular nucleoprotein particle composed of approximately equal amounts ofRibonucleic acid(RNA, 35%~55%) and protein (45%~65%).It's inside the cellprotein synthesisThe location of.Multiple ribosomes can be linked into strings to formPolyribosome。Each ribonucleosome is approximately spherical in shape, with a diameter of about 20 nm.By two differentSubunitComposition (large subunit andSmall subunit）Through magnesium ion and other bonds noncovalent bond together.It has been confirmed that there are four types of ribonucleosomes: bacteria, plants, animals and mitochondriaSubunitAnd riboribosomal RNASettlement coefficientIs different.RibosomeThere are two sites (A site and P site) that bind transfer RNA and can attachMessenger RNA，（Rb）。
Alpha Synucleinα - Synuclein is acentral nervous systemThe soluble protein expressed in presynaptic region is closely related to the pathogenesis of Parkinson's disease.α-SynucleinUnder the influence of various physiological and environmental factors, abnormal expression and aggregation, through a series of biochemical reactions such as oxidative stress, produceToxic effect, thereby participating inParkinson's diseaseOccurrence of.Understanding and studying the chemical properties, aggregation mechanism and influencing factors of α - synuclein will be very helpful for the prevention and treatment of Parkinson's disease.α - Synuclein is a soluble protein widely distributed in the brain tissue of healthy people. It is the most important protein in the pathogenesis of Parkinson's disease, because it is the main structural component of Lewy bodies. α - Synuclein aggregates andLewy body Formation andDopaminergic neuronThe death rate of is closely related.
HantavirusNucleoproteinHantavirus is caused byHemorrhagic fever with renal syndromeHantavirus, the main virus of HFRS, is composed of L, M and S segments, which respectively encode the virusRNA polymerase, envelope nucleoproteins G1 and G2 and nucleocapsid proteins (nuclear proteins for short,Nucleocapsid Protein，NP）。Serological studies show that Hantavirus nucleoprotein has strong antigenicity andImmunogenicityAt the same time, NP antigen can beSerotypeThe immune serum of Hantavirus produced cross reaction, indicating that the NP of Hantavirus has a common antigenic determinant.In view of this, we expressed and purified the core region of nucleoprotein, and usedSDS-PAGEAnd Western blottingappraisalSo as to provide technical basis for its application in diagnosis of antigen.
research meaning
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Because the nucleic acid of nucleoprotein andBiogeneticsAnd proteinbiosynthesisSo the research on the structure and function of nucleoprotein is very active.asTobacco mottle virusAndPolio virus、Influenza virusIsokineticPlant virusIt is a nucleoprotein itself, so the research of nucleoprotein is of great significance in the prevention and treatment of animal and plant diseases and clinical medicine.
Relevant knowledge
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existence
Nucleoprotein
Nucleophilic protein(karyophilic protein) refers to a class of proteins that need or can enter the nucleus to perform functions after synthesis in the cytoplasm.
Nuclear localization sequence(Nuclear localization signal, NLS): nucleophilic proteins generally contain specialamino acidSequences, these contained short peptides ensure that the entire protein can pass throughNuclear pore complexWhen transported into the nucleus, this segment of sequence with the function of "orientation" and "localization" is called nuclear localization sequence.
Nuclear output signal (NES): as the signal of the substance in the nucleus to output the nucleus, it helps some molecules in the nucleus to enter the cytoplasm quickly through the nuclear pores, which are synthesized in the nucleus and then transported to the molecules working in the cytoplasm.
loop
The process of synthesizing polypeptide chains from activated amino acids on ribosome using mRNA as template.
protein synthesisThe form refers to the combination of ribosome size subunits to form ribosomes when needed,Synthetic proteinThe process of post disintegration and reassembly to form ribosomes when needed
Ribosomal circulationRibosomal cycle: in a broad sense, ribosomal cycle refers to the process of condensation of amino acids on the ribosome to form polypeptide chains after activation. This process includes the start of peptide chain synthesis, the extension of peptide chain, the termination and release of peptide chain synthesis. In a narrow sense, ribosomal cycle refers to the peptide chain extension phase in the process of polypeptide chain synthesis, which is carried out by three steps: carry, peptide formation and transposition,Until the termination phase.
Extraction method
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Methods of extracting nucleoprotein from cells and animal tissues
Product composition 310001 310002
Specification 50 assays 100 assays
Buffer A (Cytoplasmic Extraction Reagent) 10ml 20ml
1. Take 5-10 × 106 cells, centrifuge them for 2-3 minutes under the condition of 4 ℃ and 500 × g, carefully absorb the culture medium, dry it as far as possible, and collect the cells.
2. Wash the cells twice with cold PBS, and absorb the supernatant as much as possible after each washing.
3. Add 200 μ l of cold Buffer A, 2ul protease inhibitor mixture into every 20ul cell volume, vortex at high speed for 15 seconds, and place on ice for 10 minutes.
4. High speed vortex oscillation for 5 seconds again, and then centrifugation at 4 ℃ and 16000 × g for 5 minutes.
5. Quickly inhale the supernatant into another pre cooled clean centrifuge tube to obtain cytoplasmic protein. Please put it on ice or - 80 ℃ refrigerator for standby.
6. Add 200 μ l cold Buffer B, 2ul, into the precipitationProtease inhibitorMixture, high-speed vortex oscillation for 15 seconds.
7. Put it on ice for 40 minutes, and high-speed vortex oscillates for 15 seconds every 10 minutes.
8. Centrifuge at 4 ℃ and 16000 × g for 10 minutes.
9. Quickly inhale the supernatant into another pre cooled clean centrifuge tube to obtain nucleoprotein.
B. Tissue protein extraction
1. Take an appropriate amount of tissue sample and cut it into pieces, add cold PBS, homogenize it with a tissue homogenizer until there is no obvious visible solid (or grind it with liquid nitrogen), leave it on ice for 5 minutes, and carefully inhale the supernatant into another pre cooled clean centrifuge tube.
2. Centrifuge at 4 ℃ and 500 × g for 2-3 minutes, and discard the supernatant.
3. Follow the third step of the cell protein extraction method to proceed.
The above protein extracts should be stored separately at - 80 ℃ in the refrigerator for future use.
matters needing attention:
1. All BestBio Bebo during the experimentBiological reagentPrecooling is required;All appliances must be pre cooled in a - 20 ℃ refrigerator.
2. Keep the sample at low temperature throughout the process.
