阅读说明：本技术 一种caii的突变体及其应用 (CAII mutant and application thereof ) 是由 杨艳 于 2020-04-18 设计创作，主要内容包括：本发明公开了一种CAII的突变体及其应用,本发明通过对CAII缺陷综合征的患者进行外显子测序,发现在368位的核苷酸处存在一个突变,该突变的发现为CAII缺陷综合征的诊断以及CAII相关的疾病的诊断和药物的开发提供了新手段。(The invention discloses a CAII mutant and application thereof, and the invention discovers that a mutation exists at a 368 bit nucleotide position by sequencing exons of patients with CAII deficiency syndrome, and the discovery of the mutation provides a new means for diagnosing the CAII deficiency syndrome and CAII related diseases and developing medicaments.)

1. A mutant CAII protein, characterized in that the amino acid sequence thereof is shown as SEQ ID NO.1, and has p.W123X mutation.

2. A nucleotide sequence encoding the mutated CAII protein of claim 1, wherein the nucleotide sequence is as shown in SEQ ID No.2, having the c.g368a mutation.

3. A recombinant vector comprising the nucleotide of claim 2.

4. A recombinant cell or microorganism into which the nucleotide of claim 2 or the recombinant vector of claim 3 has been introduced.

5. The primer or probe for detecting CAII gene mutation is characterized in that the mutation is c.G368A mutation.

6. Reagent for detecting a mutation in a CAII protein, wherein said mutation is a p.w123x mutation.

7. A product for detecting CAII gene or protein mutation, which comprises the primer or probe of claim 5, or the reagent of claim 6.

8. The product of claim 7, wherein the product comprises a kit, chip or formulation.

9. The device for detecting CAII gene mutation is characterized by comprising a PCR mixed solution preparation unit, a PCR reaction unit and an information processing unit, wherein the PCR mixed solution preparation unit is configured and used for mixing a DNA template to be detected, upstream and downstream PCR amplification primers aiming at a target region and a PCR premixed solution to prepare a PCR mixed solution.

10. Use according to any one of the following:

1) use of the protein of claim 1/the nucleotide of claim 2/the recombinant vector of claim 3/the recombinant cell or microorganism of claim 4/the probe or primer of claim 5/the reagent of claim 6/the product of claim 7 or 8/the device of claim 9 for the preparation of a means for diagnosing CAII deficiency syndrome;

2) use of the protein of claim 1/the nucleotide of claim 2/the recombinant vector of claim 3/the recombinant cell or microorganism of claim 4/the probe or primer of claim 5/the reagent of claim 6/the product of claim 7 or 8/the device of claim 9 for the preparation of a means for diagnosing a CAII-related disease.

Technical Field

The invention relates to the field of biological medicine, and particularly relates to a CAII mutant and application thereof.

Background

Carbonic Anhydrases (CA) are a class of zinc enzymes that catalyze the reversible reaction of carbon dioxide and water to produce bicarbonate and hydrogen ions. It is widely found in animals, plants and microorganisms and plays an important role in bioprocessing. Due to its properties, carbonic anhydrase is being widely used in applications such as biological detection, screening of natural active substances, biosensors, and CO₂Trapping and physiological diagnosis. Carbonic anhydrase has different functions in each organ: HCO located in lung erythrocytes₃ ^-And H⁺Binding to form H₂CO₃Carbonic anhydrase I, carbonic acidAnhydrase II acceleration of H₂CO₃Decomposition into CO₂And H₂Rate of O, CO₂By diffusion into the plasma, in order to replenish the HCO consumed₃ ^-HCO in plasma₃ ^-Into the red blood cells. A large amount of CA is present in parietal cells of the stomach, which mediate the synthesis and breakdown of hydrochloric acid. Recent studies have found that a decrease in mechanical force of contraction of papillary muscles is closely associated with enzyme inhibitors, and if severe, intracellular acidosis can occur. The intensive understanding of carbonic anhydrase structure and function has led researchers to find that it catalyzes CO removal₂Of reversible hydration reactions of (a), other functions: participate in ester hydrolysis reactions, catalyze hydration of acetaldehyde with other aldehydes, and the like.

CAII deficiency syndrome is caused by a mutation in the CAII gene resulting in a decrease in the activity of the encoded CAII or an encoding of an incomplete, inactivated CAII, which belongs to the autosomal recessive inheritance. Patients often exhibit bone sclerosis (Osteopetrosis), Renal Tubular Acidosis (RTA) and brain calcification (cerebral calcium). The gene coding CAII is located on chromosome 8, the total length is about 20kb, 7 exons, and the total length of CAII protein is 260 amino acids. CAII is the highest activity of CA in human body, has expression in multiple organs and tissues, and is the most studied CA in human body.

The mutation of the exon region of the gene is closely related to the occurrence and development of diseases, wherein missense mutation can directly cause the change of an amino acid sequence, thereby influencing the structure and the function of the protein. More and more researches report that the synonymous mutation without changing the amino acid sequence can also be a risk factor for disease pathogenesis, the mutation causes the splicing abnormality of the precursor mRNA to be a main pathogenesis, and it is estimated that 90 percent of disease related synonymous mutations can influence the splicing function. Pathogenic studies of synonymous mutations suggest that the pathogenic mechanism of missense or nonsense mutations is not limited to single amino acid changes at the protein level and may be associated with aberrant splicing of precursor mrnas. The exon regions of the gene contain many splicing enhancers (ESEs) and splicing silencers (ESSs), which can enhance or weaken the splicing function of the precursor mRNA. Splicing enhancers are predominantly present in the exon regions, so that mutations occurring in the coding region influence, on the one hand, the encoded protein, e.g.nonsense or missense mutations, and, on the other hand, possibly the RNA splicing by altering the splicing regulatory sequences, with more serious consequences. The detection of CAII function-related mutation through exon sequencing is of great significance for the research of CAII deficiency syndrome or CAII-related diseases.

Based on clinical research, the invention collects samples of CAII deficiency syndrome patients to carry out exon sequencing, discovers that mutation c.G368A (p.W123X) exists in the No. 4 exon of CAII, and further experiments prove that the mutation is related to the function of protein, thereby providing a molecular basis for research of disease pathogenesis and clinical application.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention aims to provide a novel mutation of CAII and application thereof.

The first aspect of the invention provides a mutant CAII protein, the amino acid sequence of which is shown in SEQ ID NO.1 and has p.W123X mutation.

The second aspect of the invention provides the nucleotide of the mutant CAII protein of the first aspect of the invention, wherein the nucleotide sequence is shown as SEQ ID NO.2, and has c.G368A mutation.

In a third aspect, the present invention provides a recombinant vector comprising a nucleotide according to the second aspect of the present invention.

In a fourth aspect, the present invention provides a recombinant cell or microorganism into which a nucleotide according to the second aspect of the present invention or a recombinant vector according to the third aspect of the present invention has been introduced.

The fifth aspect of the invention provides a primer or a probe for detecting CAII gene mutation, wherein the mutation is c.G368A mutation.

Further, the primer pair can specifically amplify a nucleic acid amplification product containing the A with the mutation at position 368.

Further, the probe can specifically bind to the nucleic acid fragment of A with mutation at position 368.

A sixth aspect of the invention provides a reagent for detecting a mutation in a CAII protein, said mutation being a p.w123x mutation.

Further, the agent may specifically bind to a protein containing a mutation at position 123.

The seventh aspect of the invention provides a product for detecting CAII gene or protein mutation, which comprises the primer or probe of the fifth aspect of the invention, or the reagent of the sixth aspect of the invention.

Further, the product includes a kit, chip or formulation.

The eighth aspect of the invention provides a device for detecting CAII gene mutation, which comprises a PCR mixed solution preparation unit, a PCR reaction unit and an information processing unit, wherein the PCR mixed solution preparation unit is configured and used for mixing a DNA template to be detected, upstream and downstream PCR amplification primers aiming at a target region and a PCR premixed solution to prepare a PCR mixed solution.

A ninth aspect of the invention provides the use of any one of:

1) use of a protein according to the first aspect of the invention/a nucleotide according to the second aspect of the invention/a recombinant vector or microorganism according to the third aspect of the invention/a probe or primer according to the fifth aspect of the invention/an agent according to the sixth aspect of the invention in/a product according to the seventh aspect of the invention/an apparatus according to the eighth aspect of the invention for the preparation of a means for diagnosing cai deficiency syndrome;

2) use of a protein according to the first aspect of the invention/a nucleotide according to the second aspect of the invention/a recombinant vector according to the third aspect of the invention/a recombinant cell or microorganism according to the fourth aspect of the invention/a probe or primer according to the fifth aspect of the invention/a reagent according to the sixth aspect of the invention in/a product according to the seventh aspect of the invention/a device according to the eighth aspect of the invention for the manufacture of a means for the diagnosis of a CAII-related disease.

Detailed Description

In the present invention, "sample" refers to a biological sample obtained from an individual for the purpose of in vitro assessment, and a sample or patient sample in the present invention may include any body fluid. Preferred samples are blood, such as serum, plasma or whole blood.

In the present invention, the term "vector" refers to a DNA product containing a DNA sequence operably linked to control sequences capable of expressing the DNA in a suitable host.A vector may be a plasmid, a phage particle, or a simple latent genomic insert.

Useful expression control sequences include, for example, the early and late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC or TRC system, the T3 and T7 promoters, the major operator and promoter regions of phage lambda, the control regions of the fd code protein, the promoters of 3-phosphoglycerate kinase or other ethylene glycol lyase, the promoters of phosphatases (e.g., Pho5), the promoters of the yeast α -mating system and other sequences known to have the structure and induction of controlling gene expression in prokaryotic or eukaryotic cells or viruses thereof, and various combinations thereof.T 7RNA polymerase promoter Φ 10 can be used to express proteins in E.coli.

As is well known in the art, in order to increase the expression level of a transgene in a host cell, the gene should be operably linked to transcriptional/translational expression control sequences that are functional in the chosen expression host. Preferably, the expression control sequences and corresponding genes are contained in a recombinant vector containing a bacterial selection marker and an origin of replication. When the expression host is a eukaryotic cell, the recombinant vector should further include an expression marker useful in eukaryotic expression hosts.

Suitable expression vectors for eukaryotic hosts include, for example, expression control sequences derived from SV40, bovine papilloma virus, adenovirus, adeno-associated virus, cytomegalovirus and retrovirus, expression vectors useful for bacterial hosts include bacterial plasmids, exemplified by those obtained from E.coli, such as pBeLucript, pGEX2T, pUC vectors, colE1, pCR1, pBR322, pMB9 and their derivatives, plasmids with a broad host range, such as RP4, phage DNA, which can be exemplified by a variety of phage lambda derivatives, such as lambda gt10, lambda gt11 and NM989, and other DNA phages, such as M13 and filamentous single-stranded DNA phages, expression vectors useful for yeast cells include 2. mu. mu.and their derivatives, vector useful for insect cells is pV L941.

Host cells transfected or transformed with the above recombinant vectors constitute another aspect of the invention. The term "transfection" as used herein refers to the introduction of DNA into a host and the replication of the DNA by extrachromosomal elements or chromosomal integration. As used herein, the term "transformation" refers to the accommodation of an expression vector by a host cell, whether or not any coding sequence is actually expressed.

The host cell of the invention may be a prokaryotic or eukaryotic cell. In addition, a host having high DNA introduction efficiency and high expression efficiency of the introduced DNA is generally used. Examples of host cells that can be used include well-known eukaryotic and prokaryotic hosts such as E.coli, Pseudomonas, Bacillus, Streptomyces, fungi, and yeast; insect cells, such as Spodoptera frugiperda (SF 9); animal cells such as CHO and mouse cells; african green monkey cells such as COS1, COS7, BSC1, BSC40 and BMT 10; and human cells in tissue culture. When a cDNA encoding the protein of the present invention is cloned, it is preferable to use a bacterial cell as a host. In the present invention, Escherichia coli is exemplified, but the present invention is not limited thereto.

It will be appreciated that not all vectors will perform the same function when expressing the DNA sequences of the present invention. Likewise, not all hosts perform the same function for the same expression system. However, one skilled in the art will be able to make appropriate selections from a variety of vectors, expression control sequences, and hosts without undue experimentation and without departing from the scope of the invention. For example, the choice of the vector should be made with regard to the host, since the vector should replicate in it. The number of replications of the vector, the ability to control the number of replications, should also be taken into account for the expression of other proteins encoded by the corresponding vector, for example the expression of antibiotic markers. In selecting expression control sequences, a number of factors should be considered. For example, the relative strength, controllability of the sequence and compatibility with the DNA sequences of the invention should be considered, especially with respect to possible secondary structures. The unicellular host can be selected taking into account factors such as the vector chosen, the toxicity of the product encoded by the DNA sequence of the invention, the secretion characteristics, the ability to fold the protein accurately, culture and fermentation factors, and the ease of purification of the product encoded by the DNA sequence of the invention. Within the scope of these factors, the skilled person can select various vector/expression control sequence/host combinations capable of expressing the DNA sequences of the invention in fermentation or in large scale culture. As a method for screening cDNA for cloning a protein by expression cloning, a binding method, a membrane emulsion method, or the like can be used.

In the present invention, the term "exon" refers to the portion of mature mRNA that is retained, i.e., the portion of mature mRNA corresponding to the gene. Introns are the parts that are spliced out during mRNA processing and are not present in mature mRNA. Both exons and introns are for genes, the encoded part is an exon, the non-encoded part is an intron, and the intron has no genetic effect.

The term "primer" refers to a generic term for oligonucleotides that can be complementary to a template and that, upon the action of a DNA polymerase, synthesize a DNA strand complementary to the template.A primer can be natural RNA, DNA, or any form of natural nucleotide, and can even be a non-natural nucleotide such as L NA or ZNA.

The term "probe" generally refers to a polynucleotide probe that is capable of binding to another polynucleotide (often referred to as a "target polynucleotide") by complementary base pairing. Depending on the stringency of the hybridization conditions, a probe can bind to a target polynucleotide that lacks complete sequence complementarity to the probe. The probe may be directly or indirectly labeled, and includes within its scope a primer. Hybridization modalities, including (but not limited to): solution phase, solid phase, mixed phase or in situ hybridization assays.

These probes may be DNA or RNA, or may be polynucleotides obtained by replacing some or all of the nucleotides with an artificial Nucleic Acid such as PNA (peptide Nucleic Acid), L NA (registered trademark), locked Nucleic Acid, ENA (registered trademark, 2 '-O, 4' -C-Ethylene-Bridged Nucleic Acid), GNA (Glycerol Nucleic Acid), TNA (Threose Nucleic Acid ) or the like

The terms "chip", "array", "biochip" are used interchangeably and refer to a collection of a plurality of probes, labels arranged on a common substrate, which may be a silicon wafer, nylon tape, plastic tape, or glass slide.

An "array," "macroarray," or "microarray" refers to an intentionally created collection of substances (such as molecules, markers, openings, microcoils, detectors, and/or sensors) that are attached to or fabricated on a substrate or solid surface (such as glass, plastic, silicon wafer, or other material forming an array). Arrays can be used to measure the level of a large number (e.g., tens, thousands, or millions) of reactions or combinations simultaneously. The array may also contain a small amount of material, for example one, a few or a dozen. The substances in the array may be the same or different from each other. The array may take a variety of forms, such as a library of soluble molecules, a library of immobilized antibodies, a library of compounds tethered to resin beads, silicon chips, or other solid supports. The array may be a macro-array or a micro-array depending on the size of the pads on the array. Macroarrays typically contain pad sizes of about 300 microns or more and can be easily imaged by gel and print scanners. Microarrays will typically contain pad sizes of less than 300 microns.

"solid support" refers to an insoluble, functionalized, polymeric material to which library members or reagents can be attached or covalently bound (often via a linker) to immobilize or allow them to be readily separated (by filtration, centrifugation, washing, etc.) from excess reagents, soluble reaction byproducts, or solvents.

In the present invention, the kit comprises one or more sterile containers, and such containers may be in the form of a box, ampoule, bottle, vial, tube, bag, sachet, blister pack, or other suitable container known in the art. Such containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for containing medicaments.

The term "DNA library" refers to a mixture of DNA fragments of a certain size obtained by disrupting a desired fragment of a genome.

Methods for preparing DNA libraries are well known to those skilled in the art and include, but are not limited to, the steps of:

1. providing a sample to be tested, said sample containing broken double-stranded nucleic acid fragments derived from genomic DNA and said nucleic acid fragments having blunt ends;

2. adding an adaptor connecting sequence at the end of the double-stranded nucleic acid fragment; adding adaptors to both ends of the double-stranded nucleic acid fragment through the adaptor-joining sequence, wherein the adaptors have a primer binding region and a junction-complementary region, the junction-complementary region being complementary to the adaptor-joining sequence; the sequence of the primer binding region of the linker flanking the 3 'and 5' ends is different.

3. Amplifying the DNA double-stranded nucleic acid fragment with the adaptor obtained in the previous step with a first primer and a second primer, thereby obtaining a mixture of PCR amplification products, wherein the primers have an adaptor binding region corresponding to the primer binding region of the adaptor and a sequencing probe binding region located outside the adaptor binding region.

In a preferred embodiment, the cleavage product, the end-repair product, the linker product and the enrichment product may also be purified. Purification conditions and parameters are well known to those skilled in the art, and it is within the ability of those skilled in the art to make certain changes or optimizations to the reaction conditions.

The terms "exon capture" and "chip hybridization" are used interchangeably and refer to the process by which a probe specifically selects and binds to DNA fragments in the exon regions of a library.

DNA molecules are normally double stranded and therefore, prior to capture, the DNA molecule must become single stranded, typically by denaturing it by heating for melting purposes, and the melted DNA molecule is rapidly cooled, i.e., remains single stranded. The library is denatured and then subjected to capture hybridization with the chip on the hybridization platform. Molecular hybridization is carried out under stringent conditions between the DNA fragments containing the exon regions and the probes immobilized on the chip. Preferably, the concentration of probe molecules on the chip is much higher than the concentration of target molecules. After hybridization, the captured sequences are collected by methods such as denaturation and purified to obtain a mixture of sequences from the captured sequences.

The present invention may utilize any method known in the art for detecting genes. It will be appreciated by those skilled in the art that the means by which the gene is detected is not an important aspect of the present invention. The genes of the present invention are detected using a variety of detection techniques known to those of ordinary skill in the art, including, but not limited to: nucleic acid sequencing, nucleic acid hybridization, nucleic acid amplification technology and immunodetection technology.

Has the advantages that:

the invention discovers the pathogenic mutation of CAII for the first time, can be used for diagnosing diseases and provides scientific basis for developing therapeutic drugs for diseases.

Meanwhile, the invention provides a CAII deficiency syndrome or CAII-related disease diagnosis product and means, which are beneficial to disease discovery.

Drawings

FIG. 1 is a family atlas of a patient;

FIG. 2 is a sanger sequencing diagram;

FIG. 3 is an immunofluorescence assay;

FIG. 4 is a Western Blot assay.

Detailed Description

The following examples are intended to illustrate the invention in further detail with reference to the accompanying drawings and examples, and are not intended to limit the scope of the invention the experimental procedures, for which specific conditions are not indicated in the examples, are generally performed according to conventional conditions, such as those described in Sambrook et al, molecular cloning, A laboratory Manual (New York: Cold Spring Harbor L aboratoryPress,1989), or according to the manufacturer's recommendations.