46 results about "Beta globin" patented technology

The invention discloses a kit for simultaneously detecting thalassemia mutant type and deletion type and application thereof, wherein the kit includes a plurality of amplimer groups for amplifying thalassemia genes; the amplimer groups include primers designed by aiming at alpha globin genes and beta globin gene, and mutant type detecting primers aiming at HBA1 genes, HBA2 genes and HBB genes, anddeletion type detection primers aiming at alpha thalassemia genes -alpha 3.7, -alpha4.2,--SEA,--THAI and beta deletion type SEA-HPFH, sinotype and Taiwantype. Through adopting the kit, the thalassemia mutant type and deletion type can be simultaneously detected, particularly, seven common deletion types and more than 300 mutant types can be simultaneously detected, and novel mutant types can be found out; furthermore, the cost is low, and the kit is much superior to the conventional thalassemia detection kit.

The invention relates to a method and kit for detecting alpha and beta thalassemia point mutation based on a next generation sequencing technology .In the detection method, PCR (polymerase chain reaction) amplification primer sequences SEQ ID NO1-17 in exon, regulation and transcription regions of HBA1, HBA2 and HBB genes, PCR marker primer sequences SEQ ID NO18-115 at the 5'-terminal and PCR marker primer sequences SEQ ID NO116-213 at the 3'-terminal involved with alpha and beta thalassemia are included. The detection method comprises the following detection steps: (1) constructing a next generation sequencing library; (2) purifying the next generation sequencing library; (3) sequencing through a next generation sequencer; and (4) performing bioinformatic analysis to obtain a result. The kit for detection comprises the above-mentioned primer sequences and the next generation sequencer solexa. The detection method provided by the invention has the characteristics of simple operation steps, high detection specificity, wide detection range, low cost and the like.

The invention discloses a beta-globin recombinant lentiviral vector. A gene sequence of the vector comprises a modified beta-globin full-length gene, a beta-globin gene control region HS2-HS3, a beta-globin gene 5' promoter, a beta-globin gene 3' enhancer, a chromatin insulator CTCF and pSIN4-CMV-K2M plasmids; and a nucleotide sequence is as shown in SEQ ID No.1. The invention further discloses an application of the recombinant lentiviral vector in transfection of hematopoietic stem/progenitor cells to obtain normal function beta-globin. An experiment proves that specific, efficient and stable expression of the lentiviral vector in erythroid cells can be achieved by using a vector expression system, pseudoviral particles with relatively high virus titer are obtained and the normal function beta-globin is obtained after transfection of the hematopoietic stem/progenitor cells.

The invention provides a kit and method for detecting mutation of a thalassemia-related gene and a use thereof and relates to the technical field of medical genetics. The kit for detection is used for detecting many types of patients such as people needing premarital checkup, people in gestation, newborns, people in a zone having high incidence of thalassemia and people having thalassemia family heredity history, can realize accurate, comprehensive, visual and simple detection of mutation of a thalassemia-related gene, has a detection mutation range comprising HBA1, HBA2 and HBB genes in the genome and neighbouring zones and has the characteristics of simpleness, accuracy, good repeatability and promotion and use easiness.

The invention relates to a recombinant adeno-associated virus vector. The recombinant adeno-associated virus vector comprises constitutively active human Nrf2 (Nrf2 (CA)), a CMV promoter/enhancer, beta-globin intron for increasing gene expression, human growth hormone poly (A) tail sequence, etc. The invention also relates to a preparation method of the recombinant adeno-associated virus vector and the application of the recombinant adeno-associated virus vector or a composition containing the recombinant adeno-associated virus vector in preparing a medicine for treating glaucoma retinal ganglion cell denaturation. The recombinant adeno-associated virus vector provided by the invention can obviously reduce the intraocular pressure of a mouse, increase the survival rate of RGCs of the mouse, and effectively treat glaucoma retinal ganglion cell pathological changes.

The invention discloses a human MTHFR and MTRR gene polymorphism detection primer, probe, test kit and method and belongs to the technical field of in-vitro nucleic acid detection. The two most common single nucleotide polymorphisms 677C>T and 1298A>C of the MTHFR and the common mutation site 66A>G of the MTHFR are designed for qualitative detection, and a specific primer containing reference genes beta globin and a probe are designed. The test kit includes a detection primer and probe combination, a reference substance, a PCR reaction liquid and the like. Three independent multiplex PCR reactions are conducted for qualitative detection for the three gene polymorphism sites, namely, MTHFR677C>T, MTHFR1298A>C, and MTHFR 66A>G respectively. The detection primer, the probe, the test kit and the method are strong in specificity, high in sensitivity, simple, rapid and convenient for large-scale application and popularization.

The orphan nuclear receptors TR2 and TR4 together constitute the DNA binding core of the 540 kDa DRED complex, a putative repressor of the human embryonic ε- and fetal γ-globin genes. Here the functional consequences of TR2 and TR4 germ line loss of function were examined, transgenic gain of function and dominant negative gain of function on human and murine β-type globin gene expression throughout development. ε-globin transcription responded in a manner consistent with the hypothesis that TR2/TR4 is a constitutive erythroid ε-globin repressor. In contrast, parallel experiments show that TR2/TR4 is a definitive stage-selective γ-globin repressor. This developmental stage-specific, gene-selective repression of the ε- and γ-globin genes by TR2/TR4 establishes, when considered in concert with the competition hypothesis, a coherent molecular rationale for hemoglobin switching (temporally specific, sequential activation of all the β-type globin genes) during vertebrate development.

The invention discloses a hematopoietic stem cell gene modification method for targeting a hemoglobin HBB mutant gene. The method 1 comprises the following steps: electro-transforming gRNA1, gRNA2 andthe mRNA of spCas9-2.0 into CD34+ cells containing the hemoglobin HBB mutant gene; and culturing the cells for one day, and transferring rAAV-HBB into the CD34+ cells, and harvesting the cells. The method 2 comprises the following steps: mixing and recombining a Cas9 protein with the gRNA1 and the gRNA2, and electro-transforming the recombinant Cas9 protein into the CD34+ cells; and culturing thecells for one day, and transferring the rAAV-HBB into the CD34+ cells, and harvesting the cells. The CD41/42 (-CTTT) in the most common HBB mutation in Guangxi province and Guangdong province in China is targeted, no other mutation or insertion sequences are left in chromosomes after gene editing is completed, and the gene editing efficiency can reach 15-20%.

The invention provides a basic group editing method for specifically repairing HBB gene mutation in a human genital system. The method comprises the steps of delivering a basic group editing system used for specifically repairing HBB gene mutation so that the basic group editing system is close to relevant sequences of HBB mutant genes, and obtaining repaired HBB genes, wherein the basic group editing system comprises a basic group editing enzyme and gRNA, the basic group editing enzyme is fusion protein, and the fusion protein includes effect protein structural domains, cytidine deaminase structural domains and uracil DNA glycosylase inhibitor structural domains of a CRISPR/Cas system. The gene editing system is guided into a human germ cell, a human fertilized ovum or a human embryo, A>Gpathogenic mutation can be precisely repaired, and thus beta-mediterranean anemia is cured. The basic group editing method has a wide application prospect in the field of genetic therapy.

The invention provides a method for detecting HBB gene mutation and HLA genotyping based on the high throughput sequencing technology and a corresponding reagent kit. An adopted primer composition comprises a primer of closely-linked single nucleotide polymorphisms (SNP) within the 1 Mb range of the up stream and the down stream of the specific amplification human embryo beta-thalassemia HBB gene and primers of the closely-linked single nucleotide polymorphisms (SNP) within the ranges at the up stream of the LHA-A gene, between the HLA-A gene and the HLA-B gene, between the HLA-B gene and the HLA-DRA gene, between the HLA-DRA gene and the HLA-DQB1 gene and at the downstream of the HLA-DQB1 gene of the specific amplification human leucocyte antigen system. The method has the advantages of university, single nucleotide polymorphisms (SNP) sequencing, high throughput, low cost, high flexibility and strong specificity.

The invention relates to a system for detecting multiple quantitative and virulent genes of H.pylori as well as kits and applications of the system. The system for detecting multiple quantitative and virulent genes of H.pylori comprises multiple pairs of primers, each for strain identification genes (16S rRNA), quantitative analysis genes ureCand Beta-globin, as well as virulent genes (cagA, vacA-s1, vacA-s2, vacA-m1, vacA-m2, iceA1, iceA2, dupA, oipA and luxS). The system for detecting multiple quantitative and virulent genes of H.pylori and the kits of the system can directly perform synchronous detection and analysis on the strain identification, quantification and virulence of a tissue sample in a same reaction system without adopting a conventional culture step or other steps, remedy defects that a conventional detection method is low in throughput, time-consuming and low in detection rate, obviously improve the accuracy of detection results, immediately provide a comprehensive, accurate and low-cost etiological diagnosis for clinic, and provide an important reference for the accurate diagnosis, differential diagnosis and disease prognosis of H.pylori infection.

The invention relates to a gene vector and a gene therapy drug for treating retina ganglion cell denaturation. The gene vector comprises dominant negative human Fas-associated protein with death domain (FADD-DN), an enhancer/promoter, a beta-globin intron capable of enhancing gene expression, a human growth hormone poly(A) tail sequence and the like. The invention further relates to application ofthe gene vector or a composition containing the gene vector to preparation of a drug for treating the retina ganglion cell denaturation. The gene vector provided by the invention has the advantages that the gene therapy vector provided by the invention can be used for treating or preventing retina ganglion cell pathological changes caused by glaucoma and the like.

The invention discloses an HBB gene kit for correcting autologous hematopoietic stem cells of a patient suffering from servious beta-thalassemia. The kit consists of a set of reagents for preparing a specific HBB-101 HIV slow virus and a set of reagents for HIV slow virus infection hematopoietic stem cells. The invention further discloses application of the kit in converting the autologous hematopoietic stem cells of the patient suffering from servious beta-thalassemia into hematopoietic stem cells with normal beta-globin synthesis functions. Experiment shows that the kit disclosed by the invention is simple to operate and relatively high in virus titer, and has wide development prospects in clinical study and treatment application, the virus infection efficiency is as high as 56.99%, and the purpose of treating the patient suffering from servious beta-thalassemia can be achieved through venous re-transfusion after PCR identification on corrected hematopoietic stem cells is implemented and the result of DNA sequencing identification is positive.

The invention discloses an HPV gene chip. The gene chip can be individually or simultaneously perform accurate detection, screening and typing on one or more HPV subtypes, can have human genome beta globin and an HPV primer amplification quality control point to monitor the whole detection process and thus being capable of having high sensitivity and specificity. The invention also discloses a preparation method of the HPV gene chip. By utilizing PCR reversal point hybridization method to screen a probe with strong specificity and optimizing the concentration of a spotting probe of the HPV gene chip, the quality and the efficiency of hybridization are improved; and in addition, the gene chip with good optimized condition is further verified with a clinical sample. The HPV gene chip provided by the invention has a better clinical application prospect.

The invention discloses a method of performing gene correction on hemopoietic stem cells of a patient with Mediterranean anemia. The method is characterized by comprising the steps of I, selecting CRISPR target near an HBB gene-28 (A/G) site, and designing a gRNA sequence; II, constructing a template plasmid, and inserting a dual-ITR (inverted terminal repeat) sequence of piggyBac between upstreamand downstream 500bp sequences of the HBB gene CRISPR target; III, co-transfecting the hemopoietic stem cells of the patient with Mediterranean anemia through a transfection reagent, the template plasmid, gRNA and Cas9 carrier, and performing puromycin screening on a cell medium; IV, authenticating and verifying a resistant gene. The CRISPR/Cas9 system and piggyBac are jointly utilized; the hemopoietic stem cells of the patient with Mediterranean anemia can be transfected effectively; homologous recombination efficiency can be improved; -28(A/G) mutation site of the patient with Mediterraneananemia can be corrected effectively.

The invention discloses a beta-globin recombinant lentiviral vector and application of the beta-globin recombinant lentiviral vector, the beta-globin recombinant lentiviral vector is pCCL-SIN-cPPT-LCR < 2.7 > K-beta-globin-Genomic-T87Q-RbPA, the beta-globin recombinant lentiviral vector comprises a pCCL-SIN-cPPT-MCS-RbPA skeleton, a 2.7 kb beta-LCR regulatory sequence and an expression cassette for expressing beta globin Hb beta A-T87Q with mutant amino acid at the 87th position, and the beta-LCR is a abbreviation of beta-globin chloride regulation. The lentiviral vector disclosed by the invention is high in packaging titer and low in vector production cost, the potential carcinogenic risk of the viral vector on tested cells is reduced, the Hb betaA-T87Q is expressed more efficiently and stably, and an important basis is provided for curing blood transfusion dependent type beta-thalassemia patients.

The invention discloses a biomarker or a biodegradation product thereof for epilepsy prediction, diagnosis, treatment and prognosis evaluation. The protein is named as Hemoglobin subunit beta, and theencoding gene of the biomarker is HBB. Particularly, the invention relates to a proteomic screening and detection method for a Hemoglobin subunit beta protein or a biodegradation product thereof. Results of the method show that before and after epilepsy primary lesion resection operation of a patient suffering from epilepsy, the contents of Hemoglobin subunit beta or biodegradation products thereof in serum are remarkably different (as shown in the figure 1 accompanying the abstract). The biomarker protein Hemoglobin subunit beta or the biodegradation product thereof disclosed by the invention has great significances in fields such as epilepsy research, prediction, diagnosis, treatment and prognosis evaluation.

The invention relates to the field of biomedical treatment, in particular to a gene expression cassette, a lentiviral vector and application of the lentiviral vector in treatment of beta thalassemia. The gene expression cassette comprises a promoter, a beta globin gene, an intron-BCL11A-shRNAmir and a polyadenylation signal which are sequentially connected; and the promoter is a type II promoter with erythroid cell specificity. The gene expression cassette can specifically up-regulate expression of beta globin and gamma globin in erythroid cells, and the two mechanisms have a synergistic effect, so that the treatment effect is remarkably enhanced.

The invention provides a siRNA sequence for targeted inhibition of human alpha-globin gene expression. The siRNA sequence is a HBA2 siRNA1 sequence and comprises a sense strand and an antisense strand; the nucleotide sequence of the sense strand is shown by SEQ ID No.1; and the nucleotide sequence of the antisense strand is shown by SEQ ID No.2. The siRNA sequence can specifically and efficiently inhibit the expression of the alpha-globin gene so as to improve the synthesis imbalance of alpha/beta globin of beta thalassemia sufferers and lay a foundation for establishing a medicine and technology system for treating beta thalassemia through siRNA. Moreover, the siRNA-mediated RNAi technology is efficient, specific and convenient and has a good application prospect.

The invention discloses a characteristic protein marker composition for screening thalassemia, a mass spectrum model and application thereof. The invention firstly discloses a characteristic protein marker composition for screening thalassemia. The composition comprises alpha globin, beta globin, delta globin, gamma globin and/or carbonic anhydrase I, and the sequences of the alpha globin, the beta globin, the delta globin, the gamma globin and/or the carbonic anhydrase I are respectively shown as SEQ ID NO.1-5. The invention further discloses a mass spectrum model containing the marker composition and application of the mass spectrum model in preparation of products for screening thalassemia. Based on the MALDI-TOF mass spectrum technology, the method can screen beta thalassemia by analyzing the mass spectrum peak areas and ratios of different characteristic proteins, carries alpha and beta thalassemia, alpha thalassemia and abnormal hemoglobin samples, and has the characteristics ofsimple sample pretreatment, high detection flux, less reagent consumables, low cost, less sampling amount, high sensitivity and the like; and large-scale population screening can be carried out in regions with high occurrence rate of thalassemia.

The invention discloses application of a lentiviral vector in preparation of a medicine for treating beta-thalassemia. The lentiviral vector is composed of a pCCL-SIN-cPPT-MCS-RbPA skeleton, a regulatory sequence A, a promoter sequence B, an enhancer sequence C and a gene sequence D. The lentivirus can enhance the specific expression of genes and reduce the size of the vector, so that the virus packaging efficiency is improved, the potential carcinogenic risk of the virus vector to tested cells is reduced, and the lentivirus vector infects hematopoietic stem cells (CD34 + cells) of beta-thalassemia patients, so that the beta-thalassemia patients are infected with hematopoietic stem cells (CD34 + cells). The expression level of exogenous beta globin (Hb beta A-T87Q) containing 87th amino acid mutation is quantified by high performance liquid chromatography, the exogenous beta globin (Hb beta A-T87Q) can be distinguished from wild beta globin expressed by an endogenous gene or derived from blood transfusion, and high expression of Hb beta A-T87Q is observed.