1.Assay of the expression and self-activition function of full length and different fragments of mayven gene in yeast two-hybrid system.
Fang LIU ; Zhiyin DU ; Jun-lin HE ; Ying-xiong WANG
Chinese Journal of Medical Genetics 2008;25(3):338-342
OBJECTIVETo identify the mayven-interacting proteins and study the effect of mayven on multiple sclerosis pathogenesis.
METHODSThe full length and four different fragments of mayven gene were amplified by polymerase chain reaction (PCR) with specific primers and were cloned into the yeast expression vector pDBLeu. Then, the recombinant plasmids were transformed into MAV203 yeast strain. The autonomous activation of their expression products was detected.
RESULTSThe yeast expression vectors of mayven, which include a full-length and four different fragments, were constructed successfully. Full length P1, fragments P3 and P4 have no effect on the expression of HIS3 and LacZ gene, but fragments P7 and P8 do. The C-terminal of Mayven gene may contain a transcription activation domain.
CONCLUSIONFull length P1, fragments P3 and P4 of the mayven gene can be used to screen the mayven-interacting proteins, but whether Mayven has transcriptional activation activity need to be studied.
Microfilament Proteins ; genetics ; Models, Genetic ; Multiple Sclerosis ; genetics ; Nerve Tissue Proteins ; genetics ; Polymerase Chain Reaction ; Two-Hybrid System Techniques
2.Prokaryotic expression and purification of different truncated protein of Mayven.
Fang LIU ; Yingxiong WANG ; Xueqing LIU ; Junlin HE
Journal of Biomedical Engineering 2008;25(6):1401-1404
To understand the function of Mayven and investigate the pathogenesis of multiple sclerosis, the gene sequences of different truncated Mayven were amplified from the gene library of human brain. These truncated fragments, including fragment P1 (1-902 bp), fragment P2 (1-523 bp), fragment P3 (507-182 bp) and fragment P4 (887-1782 bp), were cloned into pGEX-4T-2 vector to construct recombinant plasmids. The recombinant plasmids were transformed into E. coli BL21(DE3) and induced to express by IPTG. The expressed proteins were detected by SDS-PAGE and Western blot, and were purified by GST purifying system. The results showed that recombinant express vectors of different truncated GST-Mayven were successfully constructed and were expressed in soluble form protein induced by IPTG. The fusion proteins have good reactivity to GST antibody. The construction of recombinant express vectors of different truncated GST-Mayven lays a basis for further function study on Mayven.
Escherichia coli
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genetics
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metabolism
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Genetic Vectors
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Humans
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Microfilament Proteins
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genetics
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metabolism
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Multiple Sclerosis
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genetics
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Nerve Tissue Proteins
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genetics
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metabolism
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Recombinant Fusion Proteins
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genetics
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metabolism
3.Variants of Interferon Regulatory Factor 5 are Associated with Neither Neuromyelitis Optica Nor Multiple Sclerosis in the Southeastern Han Chinese Population.
Qi-Bing LIU ; Lei WU ; Gui-Xian ZHAO ; Ping-Ping CAI ; Zhen-Xin LI ; Zhi-Ying WU ;
Chinese Medical Journal 2015;128(13):1743-1747
BACKGROUNDNeuromyelitis optica (NMO) and multiple sclerosis (MS) are demyelinating disorders of the central nervous system. Interferon regulatory factor 5 (IRF5) is a common susceptibility gene to different autoimmune disorders. However, the association of IRF5 variants with NMO and MS patients has not been well studied. Therefore, we aimed to evaluate whether IRF5 variants were associated with NMO and MS in the Southeastern Han Chinese population.
METHODSFour single nucleotide polymorphisms (SNPs) were selected and genotyped by matrix-assisted laser desorption/ionization time of flight mass spectrometry in 111 NMO patients, 145 MS patients and 300 controls from Southeastern China.
RESULTSNone of these 4 SNPs was associated with NMO or MS patients.
CONCLUSIONSOur preliminary study indicates that genetic variants in IRF5 may affect neither NMO nor MS in the Southeastern Han Chinese population. Further studies with a large sample size and diverse ancestry populations are needed to clarify this issue.
Adult ; Asian Continental Ancestry Group ; genetics ; China ; Female ; Genetic Predisposition to Disease ; genetics ; Genotype ; Humans ; Interferon Regulatory Factors ; genetics ; Male ; Middle Aged ; Multiple Sclerosis ; genetics ; Neuromyelitis Optica ; genetics ; Polymorphism, Single Nucleotide ; genetics
4.Construction and identification of luciferase reporter gene containing mouse T-bet promoter.
Chinese Journal of Biotechnology 2014;30(11):1733-1741
The aim of this study is to clone the mouse T-bet promoter and enhancer, construct and identify the firefly luciferase reporter gene plasmid pGL4.10-TBX21pr-CNS for T-bet transcription regulation study and its function in signaling of multiple sclerosis. The promoter and CNS of T-bet were predicted by bioinformatics assay. The predicted fragment of mouse T-bet promoter plus CNS was amplified by PCR and cloned into pGL4.10. The recombinant plasmid pGL4.10-TBX21pr-CNS was transferred into Escherichia coli DH5α. The positive clone was identified by double digestion with Kpn I and Sfi I and DNA sequencing. Finally, pGL4.10-TBX21pr-CNS was cotransfected with pRL-TK into 293T cells and Jurkat cells, pRL-TK and pGL4.10 as a control. The luciferase activity in 293T cells (P = 0.012 2) and Jurkat cells (P = 0.002 2) was higher than that of the control group. A fragment of 1 028 bp mouse T-bet promoter plus 1 308 bp CNS was successfully cloned and the firefly luciferase reporter gene plasmid pGL4.10-TBX21pr-CNS was constructed. In 293T cells and Jurkat cells, pGL4.10-TBX21pr-CNS has the promoter functions. This work offers a basic material for the research of T-bet transcription.
Animals
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Gene Expression Regulation
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Genes, Reporter
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Genetic Vectors
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Luciferases
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Mice
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Multiple Sclerosis
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Plasmids
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Promoter Regions, Genetic
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T-Box Domain Proteins
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genetics
5.Review of neuropathology in the past 10 years in China.
Chinese Journal of Pathology 2005;34(9):550-552
6.Molecular Analysis of HLA Class ll-Associated Susceptibility to Neuroinflammatory Diseases in Korean Children.
Hyun Hee OH ; Soon Hak KWON ; Chang Woo KIM ; Byung Ho CHOE ; Cheol Woo KO ; Hee Du JUNG ; Jang Soo SUH ; Jun Hwa LEE
Journal of Korean Medical Science 2004;19(3):426-430
The work was done to study immunogenetic peculiarities of neuroinflammatory diseases among Korean children. A total of 13 children with neuroinflammatory diseases (8 males and 5 females; mean age 4.6+/-2.6 yr) were consecutively recruited. Genomic typing was performed on their HLA DRB/HLA DQB genes using PCR-SSOP/ SSP techniques with gel immunoelectrophoresis. The frequencies of HLA-DR1* 15 in children with acute disseminated encephalomyelitis (ADEM) (31%) and DQB1* 06 in other neuroinflammatory diseases (38%) were significantly increased compared with control subjects. The frequencies of HLA-DRB3*0202 (100%), HLA-DRB1*1302 (67%), HLA-DRB3*0301 (67%), and HLA-DQB1*0301 (67%) were significantly increased in children with multiple sclerosis and the frequencies of HLA-DRB1*1501 (40%) and HLA-DRB5*0101 (40%) were significantly increased in children with ADEM. HLA-DRB1*1401, HLA- DRB3*0202, and HLA-DQB1*0502 were found in children with acute necrotizing encephalopathy. In conclusion, HLA-DR1*15 and DQB1*06 may be involved in susceptibility to inflammation in Korean children. The frequencies of HLA-DRB1*1501, HLA-DRB5*0101, HLA-DRB3*0301, and HLADQB1* 0602 were not as high in Korean children with multiple sclerosis as in western children. However, HLA-DRB3*0202 was seen in all children with multiple sclerosis. Our data may provide further evidence that the immunogenetic background of neuroinflammatory diseases in Korean is distinctly different from the ones in western countries. Further studies are necessary to confirm this finding.
Alleles
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Child
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Child, Preschool
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Electrophoresis
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Encephalomyelitis/genetics
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Female
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Genes, MHC Class II/*genetics
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*Genetic Predisposition to Disease
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Genotype
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Human
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Inflammation/*genetics
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Male
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Multiple Sclerosis/genetics
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Neurons/*pathology
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Polymerase Chain Reaction
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Polymorphism, Single-Stranded Conformational
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Support, Non-U.S. Gov't
7.Deficient mRNA expression of specific protein 3 gene in peripheral blood mononuclear cells from patients with multiple sclerosis.
Ai-yu LIN ; Qi-dong YANG ; Shen-xing MURONG
Chinese Journal of Medical Genetics 2008;25(2):187-189
OBJECTIVETo characterize the deficiency of the mRNA expression of specific protein (SP3) gene in peripheral blood mononuclear cells (PBMCs) from Chinese patients with multiple sclerosis (MS) and study its correlation with the disease phenotypes.
METHODSFifty-six patients with definite MS were collected and total RNA was extracted from their PBMCs. Specific primers corresponding to SP3 gene were designed and the mRNA expression of SP3 gene was detected by reverse transcriptase-PCR (RT-PCR) method. The deficiency of SP3 expression was compared among MS patients, irrelevant disease group and normal controls.
RESULTSOf the 56 MS cases, 23 (41.1%) were SP3-deficient. In contrast, the frequency of SP3-deficiency in normal subjects and irrelevant disease controls was 8.6% (5/35) and 14.3% (4/27), respectively. The frequency of the SP3-expression deficiency in MS patients was significantly higher than that in both control groups (P< 0.01). Within the MS cases, the scores of expanded disability status scale (EDSS) in the SP3-expressing subjects were significantly different from that in the SP3-deficient ones in the stable, but not in the active, phase of MS (P< 0.05).
CONCLUSIONAuthor's observation suggested that deficient expression of SP3 gene occurs in Chinese MS patients, and that the SP3 expression may correlate with the clinical manifestations of MS and play roles in its immunological pathogenesis.
Adolescent ; Adult ; Aged ; Child ; Female ; Humans ; Leukocytes, Mononuclear ; metabolism ; Male ; Middle Aged ; Multiple Sclerosis ; genetics ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sp3 Transcription Factor ; genetics ; Young Adult
8.Association among serous and cerebrospinal fluid TNF-alpha level, gene polymorphisms of TNF-alpha and multiple sclerosis in Han nationality of southern China.
Ya-xian DONG ; Zhi-rong XU ; Pei-yu LIN
Chinese Journal of Medical Genetics 2006;23(6):677-679
OBJECTIVETo investigate the association among serous and cerebrospinal fluid (CSF) TNF-alpha level, gene polymorphisms of TNF-alpha and multiple sclerosis (MS) in Han nationality of southern China.
METHODSMS diagnosis was base on Poser (1983) criteria. Fifty-five patients with nonimmulogical diseases and 68 patients with MS from southern China were enrolled in the study, and their TNF-alpha level of serum and CSF were measured by double antibody sandwich ABC-ELISA. TNF-alpha -308G/A in 106 normal healthy subjects and 68 MS patients was genotyped with polymerase chain reaction-restriction fragment length polymorphism.
RESULTSThere was significant difference in the serous TNF-alpha level between nonimmune patients and active MS patients (234+/- 76 pg/mL vs 276+/- 71 pg/mL, P< 0.05), but not in the CSF (245+/- 83 pg/mL vs 265+/- 78 pg/mL, P> 0.05). The gene frequency distribution of TNF-alpha -308G/A was corresponding with Hardy-Weinberg equilibrium. The positive rate of genotype AA and the gene frequency of allele A of TNF-alpha were 4.4% and 14.0% in MS group, and 0 and 8.50% in healthy subjects, there was no statistical significance (P> 0.05).
CONCLUSIONThe TNF-alpha level in serum is associated with active MS, but not in the CSF. The gene polymorphisms of TNF-alpha -308G/A is not associated with MS in Han nationality of southern China.
Adolescent ; Adult ; Aged ; Asian Continental Ancestry Group ; genetics ; China ; Enzyme-Linked Immunosorbent Assay ; Female ; Gene Frequency ; Genotype ; Humans ; Male ; Middle Aged ; Multiple Sclerosis ; blood ; cerebrospinal fluid ; genetics ; Polymorphism, Genetic ; Tumor Necrosis Factor-alpha ; blood ; cerebrospinal fluid ; genetics
9.Variants of Interleukin-7/Interleukin-7 Receptor Alpha are Associated with Both Neuromyelitis Optica and Multiple Sclerosis Among Chinese Han Population in Southeastern China.
Jing-Cong ZHUANG ; Lei WU ; Mei-Zhen QIAN ; Ping-Ping CAI ; Qi-Bing LIU ; Gui-Xian ZHAO ; Zhen-Xin LI ; Zhi-Ying WU ;
Chinese Medical Journal 2015;128(22):3062-3068
BACKGROUNDNeuromyelitis optica (NMO) and multiple sclerosis (MS) are autoimmune demyelinating diseases of the central nerve system. Interleukin-7 (IL-7) and interleukin-7 receptor alpha (IL-7Rα) were proved to be important in the pathogenesis of both diseases because of the roles they played in the differentiations of autoimmune lymphocytes. The variants of both genes had been identified to be associated with MS susceptibility in Caucasian, Japanese and Korean populations. However, the association of these variants with NMO and MS has not been well studied in Chinese Southeastern Han population. Here, we aimed to evaluate the association of six IL-7 variants (rs1520333, rs1545298, rs4739140, rs6993386, rs7816065, and rs2887502) and one variant of IL-7RA (rs6897932) with NMO and MS among Chinese Han population in southeastern China.
METHODSMatrix-assisted laser desorption/ionization time of flight mass spectrometry (MassARRAY system) and Sanger sequencing were used to determine the variants of IL-7 and IL-7RA in 167 NMO patients, 159 MS patients and 479 healthy controls among Chinese Han population in southeastern China. Samples were excluded if the genotyping success rate <90%.
RESULTSStatistical differences were observed in the genotypes of IL-7 rs1520333 in MS patients and IL-7RA rs6897932 in NMO patients, compared with healthy controls (P = 0.035 and 0.034, respectively). There was a statistically significant difference in the genotypes of IL-7 rs2887502 between MS and NMO patients (P = 0.014). And there were statistically significant differences in the rs6897932 genotypes (P = 0.004) and alleles (P = 0.042) between NMO-IgG positive patients and healthy controls.
CONCLUSIONSThe study suggested that among Chinese Han population in southeastern China, the variant of IL-7RA (rs6897932) was associated with NMO especially NMO-IgG positive patients while the variant of IL-7 (rs1520333) with MS patients. And the genotypic differences of IL-7 rs2887502 between MS and NMO indicated the different genetic backgrounds of these two diseases.
Adolescent ; Adult ; Aged ; Alleles ; Asian Continental Ancestry Group ; genetics ; Child ; China ; Female ; Gene Frequency ; genetics ; Genetic Predisposition to Disease ; genetics ; Genotype ; Humans ; Interleukin-7 ; genetics ; Male ; Middle Aged ; Multiple Sclerosis ; genetics ; Neuromyelitis Optica ; genetics ; Polymorphism, Single Nucleotide ; genetics ; Receptors, Interleukin-7 ; Young Adult
10.Variant of Associated with Increasing Risk in Chinese Patients with Relapsing-remitting Multiple Sclerosis.
Sheng CHEN ; Juan ZHANG ; Qi-Bing LIU ; Jing-Cong ZHUANG ; Lei WU ; Yong-Feng XU ; Hong-Fu LI ; Zhi-Ying WU ; Bao-Gou XIAO
Chinese Medical Journal 2018;131(6):643-647
BackgroundMultiple sclerosis (MS) is a common central nervous system autoimmune disorder. Increasing number of genome-wide association study (GWAS) analyses hint that MS is strongly associated with genetics. Unfortunately, almost all the GWAS analyses were Caucasian population based. Numbers of risk loci might not be replicated in Chinese MS patients. Hence, we performed a MassArray Assay to genotype the previously reported variants located in the transcription regulation genes in order to elucidate their role in the Chinese MS patients.
MethodsOne hundred and forty-two relapsing-remitting MS (RRMS) patients and 301 healthy controls were consecutively collected from September 2, 2008, to June 7, 2013, as stage 1 subjects. Eight reported transcription regulation-related single-nucleotide polymorphisms (SNPs) were genotyped using the Sequenom MassArray system. In stage 2, another 44 RRMS patients and 200 healthy controls were consecutively collected and Sanger sequenced from April 7, 2015, to June 29, 2017, for the validation of positive results in stage 1. Differences in allele and genotype frequencies between patients and healthy controls, odds ratios, and 95% confidence intervals were calculated with the Chi-square test or Fisher's exact test. Hardy-Weinberg equilibrium was tested also using the Chi-square test.
ResultsIn stage 1 analysis, we confirmed only one previously reported risk variant, rs11129295 in EOMES gene. We found that the frequency of T/T genotype was much higher in MS group (χ = 10.251, P = 0.005) and the T allele of rs11129295 increased the risk of MS (χ = 10.022, P = 0.002). In stage 2 and combined analyses, the T allele of rs11129295 still increased the risk of MS (χ = 4.586, P = 0.030 and χ = 16.378, P = 5.19 × 10, respectively).
ConclusionsThis study enhances the knowledge that the variant of EOMES is associated with increasing risk in Chinese RRMS patients and provides a potential therapeutic target in RRMS.
Adolescent ; Adult ; Aged ; Alleles ; Asian Continental Ancestry Group ; Female ; Gene Frequency ; genetics ; Genetic Predisposition to Disease ; genetics ; Genome-Wide Association Study ; Genotype ; Humans ; Male ; Middle Aged ; Multiple Sclerosis ; genetics ; Odds Ratio ; Polymorphism, Single Nucleotide ; genetics ; T-Box Domain Proteins ; genetics ; Young Adult