WO2019037131A1 - Construction de vecteur d'expression eucaryote cd27 et préparation d'une souche cellulaire exprimant fortement cd27 - Google Patents

Construction de vecteur d'expression eucaryote cd27 et préparation d'une souche cellulaire exprimant fortement cd27 Download PDF

Info

Publication number
WO2019037131A1
WO2019037131A1 PCT/CN2017/099175 CN2017099175W WO2019037131A1 WO 2019037131 A1 WO2019037131 A1 WO 2019037131A1 CN 2017099175 W CN2017099175 W CN 2017099175W WO 2019037131 A1 WO2019037131 A1 WO 2019037131A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
plasmid
expression vector
total rna
eukaryotic expression
Prior art date
Application number
PCT/CN2017/099175
Other languages
English (en)
Chinese (zh)
Inventor
毛吉炎
Original Assignee
深圳市博奥康生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 深圳市博奥康生物科技有限公司 filed Critical 深圳市博奥康生物科技有限公司
Priority to PCT/CN2017/099175 priority Critical patent/WO2019037131A1/fr
Publication of WO2019037131A1 publication Critical patent/WO2019037131A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells

Definitions

  • the present invention relates to the construction of a eukaryotic expression vector and the use of the vector to prepare a stable expression cell line. Specifically, the present invention clones the full-length gene of CD27 into a pEGFP-N1 eukaryotic expression vector carrying a fluorescent protein tag, and transfects HeLa cells with the constructed eukaryotic expression vector to establish a Hela cell line stably overexpressing CD27.
  • CD27 belongs to the TNF receptor superfamily and is present on the surface of most T cells, B cells and NK cells. CD27 is an integral part of the formation and long-term maintenance of T cell immunity.
  • the ligand for CD27 is CD70 and plays an important role in regulating B cell activation and immunoglobulin synthesis; it mediates signal transduction and activates NF- ⁇ B and MAPK8/JNK. It plays an important role in the immunotherapy of tumors, and a lot of research is needed to achieve clinical transformation.
  • CD70 The ligand for CD27
  • CD70 plays an important role in regulating B cell activation and immunoglobulin synthesis; it mediates signal transduction and activates NF- ⁇ B and MAPK8/JNK. It plays an important role in the immunotherapy of tumors, and a lot of research is needed to achieve clinical transformation.
  • the lack of plasmids for encoding CD27 gene and CD27 overexpressing cell lines in the prior art has contributed to the progress of related research. Obstruction.
  • the present invention is based on the mRNA sequence of CD27 in GenBank, and is designed to include the upstream and downstream primers of the entire CD27 coding region.
  • the sequence of the upstream primer F1 is shown in the sequence listing SEQ NO.1, the 5' end of F1 is introduced into the EcoR I restriction site, and the downstream primer is introduced.
  • the sequence of R1 is shown in SEQ NO. 2, and the 5' of R1 is introduced into the Xma I restriction site.
  • the CD27 fragment amplification primer F2 designed for RT-PCR identification after cell transfection is shown in SEQ NO. 3 of the sequence listing, and the sequence of R2 is shown in SEQ NO. 4 of the sequence listing.
  • the present invention constructs a CD27 eukaryotic expression vector, which is prepared by the following steps: extraction of total RNA of cells expressing CD27, cloning of CD27 coding region, and construction of eukaryotic expression vector.
  • Jurkat cells were collected and added to 1 ml of Trizol lysate and allowed to stand at room temperature for 5 min.
  • RNA Reverse transcription of the total RNA obtained in the previous step using the PrimeScript RT Master Mix (Perfect Real Time) kit. Add 1 ⁇ l of total RNA and 5 ⁇ PrimeScript RT Master Mix (Perfect Real Time) 2 ⁇ l to a 200 ⁇ l reaction tube. RNase Free dH2O 7 ⁇ l, after mixing and reacting at 37 ° C for 60 min, the RNA was reverse transcribed into cDNA.
  • the pEGFP-N1 plasmid DNA and the purified CD27 coding region DNA were digested with EcoR I and Xma I, and the digested product was subjected to agarose gel electrophoresis, and the target fragment was recovered and purified, and T4 DNA ligase was added thereto, and ligated overnight at 4 °C.
  • the ligation product was transformed into competent E. coli Top 10. The monoclonals were picked and shaken in LB medium, and the plasmid was extracted for restriction enzyme digestion, and the plasmid of the target band was cut and sent to a sequencing company for sequencing.
  • the pEGFP-N1-CD27 eukaryotic expression vector obtained above is transferred into HeLa cells to obtain a cell line stably expressing CD27, and the screening and identification methods of the transfection conditions and the stable cell strain are as follows:
  • DMEM medium containing 10% fetal bovine serum was changed at 37 ° C for 4 hours in a CO 2 incubator.
  • the Hela cell line stably expressing CD27 was collected, and Hela cells transfected with Hela cells and pEGFP-N1 empty plasmid were used as controls to extract total RNA and protein of the above cells.
  • the expression of CD27 was detected from the mRNA level: the total RNA obtained from the above was reverse-transcribed to obtain cDNA, and the expression level of CD27 was detected by fluorescent quantitative PCR using primers F2 and R2 using cDNA as a template and GAPDH as an internal reference.
  • Figure 1 is a schematic diagram showing the results of quantitative PCR detection after G418 screening of cells.
  • the present invention is based on the mRNA sequence of CD27 in GenBank, and is designed to include the upstream and downstream primers of the entire CD27 coding region.
  • the sequence of the upstream primer F1 is shown in the sequence listing SEQ NO.1, the 5' end of F1 is introduced into the EcoR I restriction site, and the downstream primer is introduced.
  • the sequence of R1 is shown in SEQ NO. 2, and the 5' of R1 is introduced into the Xma I restriction site.
  • the CD27 fragment amplification primer F2 designed for RT-PCR identification after cell transfection is shown in SEQ NO. 3 of the sequence listing, and the sequence of R2 is shown in SEQ NO. 4 of the sequence listing.
  • Jurkat cells were harvested and total RNA was extracted using Trizol and reverse transcribed into cDNA. Take 1 ⁇ l of cDNA, Add 1 ⁇ l of each of the upstream and downstream primers of the CD27 coding region, 10 ⁇ l of 2 ⁇ Taq PCR Master Mix 2, and make up 20 ⁇ l with ddH 2 O.
  • the PCR reaction procedure was: 95 ° C for 5 min; 95 ° C for 20 s, 60 ° C for 20 s, 72 ° C for 60 s, 30 cycles; 72 ° C for 5 min to end the reaction.
  • the product was subjected to agarose gel electrophoresis and purified by gelation to obtain a CD27 coding region DNA.
  • the pEGFP-N1 plasmid DNA and the purified CD27 coding region DNA were digested with EcoR I and Xma I, and the digested product was subjected to agarose gel electrophoresis, and the target fragment was recovered and purified, and T4 DNA ligase was added thereto, and ligated overnight at 4 °C.
  • the ligation product was transformed into competent E. coli Top 10. The monoclonals were picked and shaken in LB medium, and the plasmid was extracted for restriction enzyme digestion, and the plasmid of the target band was cut and sent to a sequencing company for sequencing. The sequencing results also indicated that the recombinant plasmid contained the complete CD27 gene sequence.
  • the correctly sequenced bacteria were inoculated into 15 ml of LB medium (containing 100 ⁇ g/ml ampicillin), cultured at 37 ° C, 300 rpm for 16 h, and the recombinant plasmid pEGFP-N1-CD27 was extracted with Endo-Free Plasmid Mini Kit II.
  • DMEM medium containing 10% fetal bovine serum was changed at 37 ° C for 4 hours in a CO 2 incubator.
  • CD27 gene in Hela cell line stably expressing CD27 is increased by 240-fold or more, while the expression of CD27 gene in Hela cells transfected with pEGFP-N1 empty plasmid is substantially no more than that of normal Hela cells.
  • the variation indicates that the cDNA sequence of the CD27 gene provided by the present invention is successfully inserted into the pEGFP-N1 expression vector, and can specifically, stably, efficiently and stably promote the high expression of the CD27 gene.

Abstract

L'invention concerne la construction d'un vecteur d'expression eucaryote et une méthode de préparation d'une souche cellulaire à expression stable à l'aide du vecteur. Spécifiquement, la méthode comprend le clonage d'un gène pleine longueur CD27 dans un vecteur d'expression eucaryote pEGFP-N1 portant un marqueur protéique fluorescent, et la transfection de cellules Hela à l'aide du vecteur d'expression eucaryote construit pour établir une lignée cellulaire Hela surexprimant de manière stable CD27.
PCT/CN2017/099175 2017-08-25 2017-08-25 Construction de vecteur d'expression eucaryote cd27 et préparation d'une souche cellulaire exprimant fortement cd27 WO2019037131A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/099175 WO2019037131A1 (fr) 2017-08-25 2017-08-25 Construction de vecteur d'expression eucaryote cd27 et préparation d'une souche cellulaire exprimant fortement cd27

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/099175 WO2019037131A1 (fr) 2017-08-25 2017-08-25 Construction de vecteur d'expression eucaryote cd27 et préparation d'une souche cellulaire exprimant fortement cd27

Publications (1)

Publication Number Publication Date
WO2019037131A1 true WO2019037131A1 (fr) 2019-02-28

Family

ID=65439896

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/099175 WO2019037131A1 (fr) 2017-08-25 2017-08-25 Construction de vecteur d'expression eucaryote cd27 et préparation d'une souche cellulaire exprimant fortement cd27

Country Status (1)

Country Link
WO (1) WO2019037131A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112646720A (zh) * 2021-01-07 2021-04-13 广州市言康生物科技有限公司 一种针对突变型小分子抗癌药物研发装置

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102337297A (zh) * 2011-10-21 2012-02-01 南京医科大学 一种mbr-FPGS高效表达载体及其构建方法和应用
WO2013138586A1 (fr) * 2012-03-15 2013-09-19 Janssen Biotech, Inc. Anticorps anti-cd27 humains, leurs procédés et leurs utilisations

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102337297A (zh) * 2011-10-21 2012-02-01 南京医科大学 一种mbr-FPGS高效表达载体及其构建方法和应用
WO2013138586A1 (fr) * 2012-03-15 2013-09-19 Janssen Biotech, Inc. Anticorps anti-cd27 humains, leurs procédés et leurs utilisations

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
YIN YING: "The Study of the Immuneprotective Responses Induced by the HBcAg Fusion Protein Expressing DNA vaccine in the Mice Model", MEDICINE & PUBLIC HEALTH, 30 April 2011 (2011-04-30), pages E059, ISSN: 1674-022X *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112646720A (zh) * 2021-01-07 2021-04-13 广州市言康生物科技有限公司 一种针对突变型小分子抗癌药物研发装置
CN112646720B (zh) * 2021-01-07 2022-07-05 东莞兰卫医学检验实验室有限公司 一种针对突变型小分子抗癌药物研发装置

Similar Documents

Publication Publication Date Title
US9371533B2 (en) Protein expression system
CN106755089B (zh) 表达山羊淋巴细胞活化分子的细胞系及其构建方法与应用
CN106381310B (zh) 一种用t7噬菌体rna聚合酶和t7启动子系统在哺乳动物细胞表达蛋白的方法
WO2019037131A1 (fr) Construction de vecteur d'expression eucaryote cd27 et préparation d'une souche cellulaire exprimant fortement cd27
JP6960409B2 (ja) プロモーター
CN113025579A (zh) 一种稳定敲低猪abhd16a基因的ST-KDABHD16A细胞系及其构建方法
Uchida et al. Regulation of aquaporin-2 gene transcription by GATA-3
CN110938656A (zh) 重组表达大熊猫促卵泡生成素的载体、表达系统及制备方法
CN107200779B (zh) 稳定过表达LAT3的β细胞株的制备方法及其应用
CN106831975B (zh) 热休克转录因子1在调控15kDa硒蛋白表达中的应用
WO2019037134A1 (fr) Construction d'un vecteur d'expression eucaryote huata et préparation pour une souche cellulaire exprimant fortement huata
CN109320597B (zh) 狐亚科激活素a蛋白及其制备与应用
CN113201543A (zh) 企鹅珍珠贝Tyr基因的启动子及其应用
CN108753819B (zh) 真核表达载体、真核表达系统、二者的制备方法和应用及gdf11蛋白
CN112877332A (zh) 一种由双荧光素酶报告基因检测鸡ripk2启动子活性的方法
CN112877309A (zh) 一种N端延长型PTEN亚型PTENζ蛋白及其编码基因和应用
WO2019041066A1 (fr) Construction d'un vecteur d'expression eucaryote fh3 et préparation d'une souche cellulaire à haute expression à l'aide de celui-ci
CN101245345B (zh) 山羊S6K1基因cDNA编码区核苷酸序列
CN107119072B (zh) 一种过表达zeb2基因质粒及其构建方法和应用
CN113637675B (zh) 一种人血清白蛋白的生产方法、核苷酸序列、表达载体及表达系统
CN111494616A (zh) 一种新冠病毒免疫增强型基因疫苗及其制备方法
CN106701764B (zh) 15kDa硒蛋白基因的启动子及其核心区与应用
CN106075396B (zh) Cul4蛋白的应用
CN106177906B (zh) Ddb1蛋白的应用
CN105949285B (zh) Rbbp5截短体的应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17922450

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17922450

Country of ref document: EP

Kind code of ref document: A1