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Hans MBL length gene expression in CHO cells

Author: LaiQinTao
Tutor: ChenZhengLiang
School: Southern Medical University,
Course: Immunology
Keywords: Mannan -binding lectin Full-length gene Clone Restructuring Mammalian cell expression
CLC: Q987
Type: Master's thesis
Year: 2009
Downloads: 48
Quote: 0
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Abstract


Mannan binding lectin (mannan-binding lectin, MBL) belongs to mammalian C-type lectin superfamily family gelling hormone in the serum mostly oligomeric form, tetramer or higher is generally considered a high oligonucleotide mer that has the biological activity. MBL through its carbohydrate recognition domain (carbohydraterecognitiondomain, CRD) pathogen recognition surface mannose, fucose, N-acetyl glucosamine, N-acetyl glucosamine as the terminal mannose glycosylation structure of sugar, it has been shown that it can combine A Group B Streptococcus, Staphylococcus aureus, Listeria monocytogenes, Neisseria meningitidis bacteria, Chlamydia pneumoniae, influenza virus, HIV, Candida albicans, a small ball of Cryptosporidium and other pathogens, activate the lectin complement pathway and mediate phagocytosis conditioning, anti-infective natural immune response play. Thus, MBL is considered an important natural immune system of pattern recognition molecules. Low levels of serum MBL and functional defects repeated infection with multiple pathogens, women habitual abortion, autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis and other diseases, mainly caused by the defective MBL MBL gene point mutations. Has been found in MBL structural gene mutations leading three point defects can cause MBL, they are CGT52TGT, GGC54GAC and GGA57GAA, are located in the MBL gene exon 1, which three point mutations in autosomal dominant inheritance, the corresponding genotype were D, B, C type, the wild-type to A type. In the general population, MBL gene mutation frequency is very high, MBL defects become the most common one of immunodeficiency disease, thereby MBL replacement therapy is also ready. Plasma purified native MBL replacement therapy has been reported in the literature, and has passed a clinical trial, the results gratifying. But the problem is the very low content of human plasma MBL, high cost of extraction, the source is limited blood supply, while avoiding the shortcomings of blood products can not be generally such as potential HCV, HIV and other viral contamination. In addition, due to blood components used in scientific research complex and difficult to extract high purity MBL, MBL makes biological function of increasing difficulty. Therefore, a large number of in vitro preparation of structure and function similar to natural MBL recombinant protein is significant to study the regulation of gene expression MBL also significantly equally important. Our group has cloned the cDNA of Chinese Han people that MBL MBL coding sequence, and in mammalian cells for expression of this gene, but the expression efficiency and the expressed protein oligomers component owed satisfied. In view of such non-coding intron sequences have a moderating effect on protein expression, try this experiment, in vitro expression of full-length gene MBL. 1 full-length Hans MBL gene cloning and sequence analysis using mannan-coated to capture serum MBL, with anti-human MBL antibody (HYB131-11) detection ELISA system serum MBL level. Blood samples from eight patients without a history of recurrent infections and autoimmune disease with a history of adult healthy Han Chinese individuals, each sample was diluted by 1:2,1:4,1:8, three wells per dilution testing, test results are applied SPSS13. 0 software is multi-level two-factor repeated measurement methods for statistical analysis, screening out high levels of blood serum MBL used to extract leukocyte genomic DNA. As a template, primers were designed, the application of Platinum Taq High Fidelity PCR enzyme length MBL gene was amplified, using a pCR (?)-XL-TOPO TA cloning vector, the plasmid was constructed pXL-MBL. Miniprep plasmid by restriction analysis, PCR identification of evacuation Invitrogen Corporation sequenced. ELISA results showed OD 450nm values ??increase with decreasing dilution (F = 99.351, P <0.001), described detection system is reliable and different between samples OD 450nm values There are significant differences (F = 27.468, P <0.001). Select OD 450nm values ??higher leukocyte sample for extracting genomic DNA, full-length MBL gene was amplified and cloned. Sequence analysis showed that, MBL gene reverse insertion pCR (?)-XL-TOPO, length 6 321 bp, and gene sequences in GenBank (NC 0 00010.9 Region: 54195146 ~ 54201466) compared with 17 different bases: one in which only the coding region (exon 4), the MBL gene 3 of the base 195, resulting in change in codon 126 (CTC → CTG), but they are encoded by leucine, and the position of the base Jiqia person in GenBank MBL cDNA (AH006353) consistent; other bases are inconsistent in introns or non-coding exon region. This shows that we have successfully cloned the full-length A-type MBL genes, the recombinant plasmid pXL-MBL. 2.MBL eukaryotic expression plasmid according pXL-MBL, pcDNA3.1 (-) and pCI-neo different enzyme spectrum, using different enzymes, MBL gene construct recombinant eukaryotic expression plasmid. (1) pcDNA3.1 (-)-MBL Construction: with Kpn Ⅰ and Not Ⅰ were digested vector pcDNA3.1 (-) and the plasmid pXL-MBL, and the vector fragment was gel purification gene fragment using T4 ligase to connect and the product was transformed into E. coli TOP10F '. Ampicillin and screened positive for expanding culture of transformed bacteria, plasmid miniprep of the recombinant plasmid to enzyme digestion, PCR and sequencing. The results showed that A-type MBL gene is to forward incoming vector pcDNA3.1 (-), the recombinant expression plasmid pcDNA3.1 (-)-MBL. (2) pCI-MBL of construction: the first with Mlu Ⅰ and Sma Ⅰ complete digestion of plasmid pXL-MBL, then add Xho Ⅰ be incomplete digestion, the resulting fragments are 1 429 bp, 1 952 bp, 2 734 bp, 3 718 bp, 4 686 bp, 6 552 bp, 8 404 bp length difference of seven larger DNA fragments, of which 6 552 bp DNA bands shall contain Mlu Ⅰ and Xho Ⅰ digested sticky ends MBL full-length gene fragment was gel electrophoresis, the target band was cut out, the recovered DNA fragment with Mlu Ⅰ and Xho Ⅰ digested pCI-neo vector fragment obtained ligation reaction, also transformed into E. coli TOP10F '. Ampicillin positive transformants screened for the recombinant plasmids to digestion, PCR and sequencing. The results showed that A-type MBL gene is to forward incoming carrier pCI-neo, the recombinant expression plasmid pCI-MBL. 3.pcDNA3.1 (-)-MBL expression in CHO cells linearized plasmid pcDNA3.1 (-)-MBL after electroporation transfected into CHO cells, and 400μg/mL successively G418800μg/mL containing complete medium in order Screening culture 9 d and 12 d. Screened stably transfected cell colony for expanding culture, line by RT-PCR for cloning while training, the use of the aforementioned ELISA system for detection, each hole located three wells, test results SPSS13.0 software application by one-wayANOVA France and Dunett multiple comparison analysis of the cloning method of the hole and twice the negative control group, the difference between the detected value is higher than twice the negative control value were considered positive holes. The results showed positive wells were E10 (P = 0.001), F10 (P = 0.001), D8 (P <0.001). Select D8 for expanding culture, culture supernatants collected supernatant by mannan-Sepharose 4B affinity chromatography purified protein underwent Western-blot identification, initially identified with high polymer recombinant MBL expression, but the expression yield is not high. This may be unstable due to the integration of the MBL gene, cell proliferation, gene loss occurs, and the competitive growth of expression strains, resulting in the expression of recombinant proteins is not high, it may be the other way defective recombinant protein purification. These are subject to further cloning cultured clones stably expressing cell lines high, while optimizing cell culture and protein purification methods, and further study its biological characteristics and functions.

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