WO2018170711A1 - Human gp34 gene high-expression vector and application thereof - Google Patents

Abstract

Provided is a method for constructing a human GP34 gene expression vector, comprising the steps of: (1) cloning of a GP34 gene; and (2) construction of an overexpression vector pEGFP-C1/GP34.

Description

 Title: Human GP34 gene high expression vector and its application

 [0001] The present invention belongs to the field of biotechnology, and relates to a method for constructing a human GP34 gene high expression vector and an application thereof.

 Background technique

 [0002] GP34 is a member of the TNF receptor superfamily and is a type I transmembrane glycoprotein. The expression profile of GP34 is restricted to the surface of activated CD4+ and CD8+ T cells, and is predominantly CD4+ T cells. The human GP34 ligand (GP34/CD 134L) contained 183 amino acids in 1991 (extracellular 139 amino acids, transmembrane 21 amino acids, intracellular 23 amino acids), belonging to the TNF family, and is a scorpion-type transmembrane glycoprotein. IMD16/GP34 is an important co-stimulatory molecule that plays an important role in the body's immune response and various diseases. Its interaction can promote the activation, proliferation, migration, prolongation of life and promote hair growth of CD+4 T cells. The formation of the center and the differentiation and maturation of DC.

 technical problem

 [0003] GP34 can synergistically stimulate the activation of T cells, promote the production of high titer antibodies and class switching of B cells, mediate the infiltration of I MD16+ T cells into the inflammatory reaction site, and play an important role in the immunotherapy of tumors. The clinical transformation value is very large, and it requires a solid research before it can be put into practical use. However, the lack of the plasmid of the recombinant GP34 gene in the prior art has hindered the progress of related research.

 Problem solution

 Technical solution

 [0004] The object of the present invention is to overcome the deficiencies in the prior art and to provide a method for constructing a human GP34 gene expression vector. Based on the cloning of human GP34 gene cDNA sequence, the over-expression vector pEGFP-Cl/GP34 was constructed, which laid a foundation for the subsequent study of human GP34 gene function.

 [0005] The specific technical solution thereof is:

 [0006] A method for constructing a human GP34 gene overexpression vector, comprising the steps of:

[0007] (1) GP34 gene cloning

[0008] Extracting total RNA from Jurkat cells, reverse transcription into cDNA, and designing primers GP34-F and GP34-R to The cDNA was used as a template, and PCR amplification was carried out according to a conventional method using primers and PrimeStar high-fidelity DNA polymerase; the reaction conditions were: 98 ° C for 2 min; 98 ° C for 10 s, 58 ° C for 10 s, 72 ° C for 50 s, 30 cycles; 72 ° C for 5 min. The PCR amplification products were identified by agarose gel electrophoresis.

(2) Construction of overexpression vector pEGFP-Cl/GP34

 [0010] The purified PCR product and the eukaryotic expression vector pEGFP-Cl were digested with restriction enzymes Xho I and EcoR I, identified and recovered by 1% agarose gel electrophoresis; The target gene fragment was ligated with the same double-digested expression vector pEGFP-Cl; the reaction system was: 10XT4 DNA ligase Buffer 1 μί, linearized pEGFP-Cl vector 1 μί, PCR product 3 μί, T4 DNA ligase 1 Μί, dH20 4 L, 4 °C ligation overnight; the ligation product was transformed into E. coli DH5ot competent cells, ice bath for 30 min, heat shock at 42 °C for 90 s, immediately transferred to ice for 2 min, then added to 500 37 The pre-warmed non-resistant LB medium was incubated at 37 °C for 1 h. Finally, the bacterial solution was uniformly coated on LB solid medium containing kanamycin, and placed in a 37 ° C incubator for overnight culture. Positive colonies were screened; 3 monoclonal colonies were randomly picked. After the colony PCR was positive, the bacterial liquid was sent to Shanghai Biotech for sequencing. The obtained recombinant eukaryotic expression plasmid was named pEGFP-Cl/GP34.

 Advantageous effects of the invention

 Beneficial effect

 [0011] The present invention constructs the overexpression vector pEGFP-Cl/GP34 of the GP34 gene. Subsequent development of overexpression GP3

The 4 gene will play an important role in GP34-related drug research and development.

 Brief description of the drawing

 DRAWINGS

 1 is the relative level of GP34 gene of 293T cells transfected with pEGFP-C 1/GP34 vector.

 BEST MODE FOR CARRYING OUT THE INVENTION

 BEST MODE FOR CARRYING OUT THE INVENTION

[0013] The present invention will be further described below in conjunction with the drawings and specific embodiments.

[0014] Jurkat cells and 293T cells were purchased from ATCC, Premix PrimeSTAR

 HS enzyme was purchased from Takara, RNeasy Mini Kit was purchased from QIAGEN, Endo-Free Plasmid

Mini Kit II was purchased from Omega Bio-tek. [0015] Example 1 cloning of the GP34 gene

 [0016] Total RNA of Jurkat cells was extracted, reverse-transcribed into cDNA, primers (GP34-F, GP34-R) were designed, and PCR was carried out according to a conventional method using c-DNA as a template, using primers and PrimeStar high-fidelity DNA polymerase. The reaction conditions were: 98 ° C 2 min; 98. C 10 s, 58. C 10 s, 72. C 50 s, 30 cycles; 72 °C 5

 Min. The PCR amplification products were identified by agarose gel electrophoresis. The nucleotide sequence of GP34-F is shown in SEQ ID No: 1, and the nucleotide sequence of GP34-R is shown in SEQ ID No: 2.

Example 2 Construction of overexpression vector pEGFP-Cl/GP34

 [0018] The purified PCR product and the eukaryotic expression vector pEGFP-Cl were digested with restriction enzymes Xho I and EcoR I, identified and recovered by 1% agarose gel electrophoresis; The target gene fragment was ligated with the same double-digested expression vector pEGFP-Cl; the reaction system was: 10XT4 DNA ligase Buffer 1 μί, linearized pEGFP-Cl vector 1 μί, PCR product 3 μί, T4 DNA ligase 1 Μί, dH20 4 L, 4 °C ligation overnight; the ligation product was transformed into E. coli DH5ot competent cells, ice bath for 30 min, heat shock at 42 °C for 90 s, immediately transferred to ice for 2 min, then added to 500 37 The pre-warmed non-resistant LB medium was incubated at 37 °C for 1 h. Finally, the bacterial solution was uniformly coated on LB solid medium containing kanamycin, and placed in a 37 ° C incubator for overnight culture. Positive colonies were screened; 3 monoclonal colonies were randomly picked. After the colony PCR was positive, the bacterial liquid was sent to Shanghai Biotech for sequencing. The obtained recombinant eukaryotic expression plasmid was named pEGFP-Cl/GP34.

 Example 3 Transduction of 293T cells

 [0020] A large amount of Escherichia coli containing pEGFP-Cl/GP34 plasmid was cultured, and Endo-Free Plasmid Mini Kit II was used to extract pEGFP-Cl/GP34 plasmid. 293T cells with good growth were inoculated into six wells with 10000 cells per well. After 18 h of culture, pEGFP-Cl/GP34 plasmid was transduced into 293T cells with Lipofectamine 2000, and culture was continued for 48 h.

 [0021] Example 4 Fluorescence quantitative PCR detection of GP34 gene expression

 [0022] According to the GAPDH and GP34 gene mRNA sequences, the primer design software Oligo 7.0 was used to design the bow.

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 [2933] 293T cells transfected with 293T cells and transfected with pEGFP-Cl/GP34 plasmid were separately plated into 6-well plates. The cell density reached δΟ^^Ο^吋, and the total RNA of each group was extracted with RNeasy Mini Kit. The mRNA was reverse-transcribed into cDNA using PrimeScrip RT reagent Kit and stored at -20 °C.

 [0024] Take cDNA 1 of each group as a template, use GAPDH as an internal reference, and detect the relative expression of GP34 by real-time PCR. Set the reaction conditions: 95 ° C 30 s, 1 cycle, 95 ° C 10 s, 54 ° C 30 s 40 cycles . The relative expression of GP34 gene in each group was detected by SYBR Primescript RT-PCR Kit. The results are shown in Figure 1. It can be seen that the expression level of GP34 gene of 293T cells transfected with pEGFP-Cl/GP34 plasmid is 190-fold higher than that of normal 293T cells, indicating that the pEGFP-Cl/GP34 plasmid can be specific, sustained and efficient. , stably promote high expression of GP34 gene.

 The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto. Any person skilled in the art can make some simple within the technical scope disclosed by the present invention. Deduction or replacement is within the scope of the present invention.

 Industrial applicability

 The present invention constructs the overexpression vector pEGFP-Cl/GP34 of the GP34 gene. Subsequent development of overexpression GP3

The 4 gene will play an important role in GP34-related drug research and development.

Claims

Claims [Claim 1] A method for constructing a human GP34 gene expression vector, comprising the steps of:  (1) GP34 gene cloning  The total RNA of Jurkat cells was extracted and reverse transcribed into cDNA. Primers GP34-F and GP34-R were designed. Using cDNA as a template, primers and PrimeStar high-fidelity DNA polymerase were used for PCR amplification according to the conventional method. The reaction conditions were: 98°. C 2 min ; 98. C 10 s, 58. C 10 s, 72. C 50 s, 30 cycles; 72. C 5 min. The PCR amplification product was identified by agarose gel electrophoresis.  (2) Construction of overexpression vector pEGFP-Cl/GP34  Use of restriction enzymes Xho I and EcoR  I purified the PCR product and the eukaryotic expression vector pEGFP-Cl were digested and identified by 1% agarose gel electrophoresis; the recovered target gene fragment and the same double digestion The expression vector pEGFP-Cl was ligated; the reaction system was: 10xT4 DNA ligase Buffer 1 μί, linearized pEGFP-Cl vector 1 μί, PCR product 3 μί, T4 DNA ligase 1 μί, dH20 4 μί, 4 ° C overnight; The ligation product was transformed into E. coli DH5ot competent cells, incubated in ice bath for 30 min, heat shocked at 42 °C for 90 s, immediately transferred to ice for 2 min, and then added to 500 L 37 ° C pre-warmed non-resistant LB medium. After incubating at 37 °C for 1 h, finally, the bacterial solution was uniformly coated on LB solid medium containing kanamycin, placed in a 37 ° C incubator for overnight culture, and positive colonies were screened; 3 monoclonal clones were randomly picked. Colonies, colony PCR was positive, and the bacterial liquid was sent to Shanghai Biotech for sequencing; the obtained recombinant eukaryotic expression plasmid was named pEGFP-Cl/GP34.