A preparation method and application of recombinant fusion protein v12-cd137l
A technology of V12-CD137L and fusion protein, applied in the field of biomedicine, can solve the problems of no biological activity and low biological activity of CD137L protein, and achieve excellent effect, enhance the level of cellular immunity, and promote the effect of lymphocyte function
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0051] Example 1 Preparation of V12-CD137L recombinant protein
[0052] (1) Using the amino acid sequence described in SEQ ID NO.1 as a template, using the codon merger principle and the codon usage frequency of Escherichia coli, the nucleotide sequence for expressing the V12-CD137L recombinant protein was designed, and in the nucleotide A histidine tag catcaccatcatcaccac was introduced into the 5' end of the acid sequence, and the gene sequence of SEQ ID NO.2 was finally designed. The gene sequence of SEQ ID NO.2 was synthesized by artificial gene synthesis technology, and the gene sequence of SEQ ID NO.2 was inserted into pET28a E. coli expression plasmid through two restriction sites of NdeI and XbaI to obtain the recombinant plasmid SEQ ID NO.2-pET28a.
[0053] (2) Take a 1.5 ml centrifuge tube containing 50 microliters of Escherichia coli BL21 (DE3) competent cells, place the tube on ice for 5 minutes, add 10 nanograms of SEQ ID NO.2-pET28a recombinant plasmid, and mix ge...
Embodiment 2
[0067] Example 2 Effect of V12-CD137L protein on mouse lymphocyte proliferation in vitro
[0068] (1) The Babl / c mice were sacrificed, and the spleen was taken and crushed gently under sterile conditions to obtain a spleen cell suspension.
[0069] (2) The obtained spleen cell suspension was treated with erythrocyte lysate for 5 minutes, centrifuged at 1000 rpm to discard the supernatant, then washed twice with serum-free RPMI-1640 medium, and finally the cell pellet was resuspended in a certain amount of serum RPMI In -1640 medium, the mouse spleen lymphocyte suspension was obtained.
[0070] (3) Dilute the mouse spleen lymphocyte suspension to 5×10 6 cells / ml, seeded in 96-well cell culture plate, 100 μl per well.
[0071] (4) At the same time, the CD137L recombinant protein and the obtained V12-CD137L protein cell culture solution were diluted to serial concentrations of 5 mg / ml and 1 mg / ml respectively, and 1 microliter of each concentration of protein was added to the c...
Embodiment 3
[0075] Example 3 Enzyme-linked dot immunoassay to detect the in vivo immune enhancement effect of V12-CD137L protein on HIV gp120 protein
[0076] (1) Experimental animals Three Babl / c mice were set up in each group, which were divided into three groups: V12-CD137L protein group, HIV gp120 protein group, and HIV gp120 protein and V12-CD137L protein co-immunization group. Among them, the inoculation amount of HIV gp120 protein group was 10 micrograms, and the inoculation amount of V12-CD137L protein was 5 micrograms. Each mouse was boosted immunized two days apart, and inoculated three times in total.
[0077] (2) The mice were sacrificed on the 7th day of the initial immunization, and the spleen was removed and crushed gently under aseptic conditions to obtain a spleen cell suspension.
[0078] (3) The obtained spleen cell suspension was treated with red blood cell lysate for 5 minutes, centrifuged at 1000 rpm to discard the supernatant, then washed twice with serum-free RPMI-...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More