Difference between revisions of "Part:BBa K4645002"
Ruixuan Zhu (Talk | contribs) |
Ruixuan Zhu (Talk | contribs) (→Materials and Method) |
||
| Line 35: | Line 35: | ||
1.Expression and Purification | 1.Expression and Purification | ||
| − | <p> 1) Plasmid pET-28a(+)-NeuB (with His tag) is transformed to <i>Escherichia coli</i> BL21(DE3). The E. coli strain is cultured in LB medium containing 50 μg/mL kanamycin. </p> | + | <p> 1) Plasmid pET-28a(+)-NeuB (with His tag) is transformed to <i>Escherichia coli</i> BL21(DE3). The <i>E. coli</i> strain is cultured in LB medium containing 50 μg/mL kanamycin. </p> |
<p> 2) When the optical density of the cultured bacteria reached approximately 0.6, IPTG was added to the final concentration 1 mM. And the bacteria were induced at 18℃ overnight. The harvested bacteria are resuspended with a binding buffer (Sangon Biotech, Shanghai, China), and then the bacteria are lysed by ultrasonication. Purification is performed following the instructions of Ni-NTA SefinoseTM Resin (Sangon Biotech, Shanghai, China). </p> | <p> 2) When the optical density of the cultured bacteria reached approximately 0.6, IPTG was added to the final concentration 1 mM. And the bacteria were induced at 18℃ overnight. The harvested bacteria are resuspended with a binding buffer (Sangon Biotech, Shanghai, China), and then the bacteria are lysed by ultrasonication. Purification is performed following the instructions of Ni-NTA SefinoseTM Resin (Sangon Biotech, Shanghai, China). </p> | ||
| Line 43: | Line 43: | ||
<p> 1) Microtiter plates were coated overnight at 4 ℃ with FelD. </p> | <p> 1) Microtiter plates were coated overnight at 4 ℃ with FelD. </p> | ||
<p> 2) Plates were blocked with 1% BSA for 1 h at RT. </p> | <p> 2) Plates were blocked with 1% BSA for 1 h at RT. </p> | ||
| − | <p> 3) | + | <p> 3) Add 200 μl ELISA Washing Buffer to wash. </p> |
<p> 4) Different combinations of proteins(NeuA, NeuB, NeuA-NeuB, ClyR) were mixed on the microtiter plates for 90 min at 37℃. </p> | <p> 4) Different combinations of proteins(NeuA, NeuB, NeuA-NeuB, ClyR) were mixed on the microtiter plates for 90 min at 37℃. </p> | ||
| − | <p> 5) | + | <p> 5) Add 200 μl ELISA Washing Buffer to wash 2 time. </p> |
| − | <p> 6) | + | <p> 6) Incubate 100 μl Standard IgE sample (25 ng/μl) or 100 μl sera from allergic donors which was diluted 5-fold at 37℃ for 90 min. </p> |
| − | <p> 7) | + | <p> 7) Incubate 100 μl biotin-labeled IgE antibody at 37℃ for 1 h. </p> |
| − | <p> 8) | + | <p> 8) Add 350 μl ELISA Washing Buffer to wash 4 time. </p> |
| − | <p> 9) | + | <p> 9) Add 100 μl HRP-conjugated Streptavidin at 37℃ for 30 min. </p> |
| − | <p> 10) | + | <p> 10) Add 300 μl ELISA Washing Buffer to wash 4 time. </p> |
| − | <p> 11) | + | <p> 11) Add 90 μl color developer (dark) at 37℃ for 15 min. </p> |
| − | <p> 12) | + | <p> 12) Add 50 μl termination solution and measuring OD value immediately with microplate reader at 450 nm wavelength. </p> |
| + | |||
===Result=== | ===Result=== | ||
<html> | <html> | ||
Latest revision as of 15:11, 12 October 2023
NeuB: scFv ( single-chain fragment variable）that inhibits the immune response to the feline
Velocimmune mice were immunized with recombinant dimeric Fel d 1 and produced polyclonal antibodies [1]. Among the resulting polyclonal antibodies, a monoclonal antibody named REGN1909 was found to be the most effective in blocking the immune response to the recombinant dimeric Fel d 1. Through a literature review, we discovered both light chain and heavy chain genes of REGN1909. By fusing the C-terminus of the light chain with the N-terminus of the heavy chain using a rigid linker (GGGGS)3, an scFv was obtained. An OmpA signal peptide was fused to the N-terminus of the scFv to help it secrete into the periplasmic space, where it can more effectively neutralize the antigen.
Protein Molecular Structures
Design
The plasmid we designed consists of T7 promoter, lac operator, RBS, NeuB coding sequence, His tag, and T7 terminator, which are arranged in an order on a pET28 backbone. We aim to induce the transcription of the downstream NeuB by adding the IPTG to initiate the expression. The protein will then be purified and block activity to FelD was tested by blocking ELISA. We determine the binding effect by coating FelD in the microtiter plates then incubating NeuB and finally measuring the absorbance.
Materials and Method
1.Expression and Purification
1) Plasmid pET-28a(+)-NeuB (with His tag) is transformed to Escherichia coli BL21(DE3). The E. coli strain is cultured in LB medium containing 50 μg/mL kanamycin.
2) When the optical density of the cultured bacteria reached approximately 0.6, IPTG was added to the final concentration 1 mM. And the bacteria were induced at 18℃ overnight. The harvested bacteria are resuspended with a binding buffer (Sangon Biotech, Shanghai, China), and then the bacteria are lysed by ultrasonication. Purification is performed following the instructions of Ni-NTA SefinoseTM Resin (Sangon Biotech, Shanghai, China).
2.Blocking Elisa
1) Microtiter plates were coated overnight at 4 ℃ with FelD.
2) Plates were blocked with 1% BSA for 1 h at RT.
3) Add 200 μl ELISA Washing Buffer to wash.
4) Different combinations of proteins(NeuA, NeuB, NeuA-NeuB, ClyR) were mixed on the microtiter plates for 90 min at 37℃.
5) Add 200 μl ELISA Washing Buffer to wash 2 time.
6) Incubate 100 μl Standard IgE sample (25 ng/μl) or 100 μl sera from allergic donors which was diluted 5-fold at 37℃ for 90 min.
7) Incubate 100 μl biotin-labeled IgE antibody at 37℃ for 1 h.
8) Add 350 μl ELISA Washing Buffer to wash 4 time.
9) Add 100 μl HRP-conjugated Streptavidin at 37℃ for 30 min.
10) Add 300 μl ELISA Washing Buffer to wash 4 time.
11) Add 90 μl color developer (dark) at 37℃ for 15 min.
12) Add 50 μl termination solution and measuring OD value immediately with microplate reader at 450 nm wavelength.
Result
The molecular weight of NeuB is around 28.26 kDa. Thus, the result of SDS-PAGE above indicating that we had the expression of NeuB in our chassis E. coli BL21(DE3) (Figure 3).
Then we successfully expressed and purified the NeuB in the E. coli (Figure 4).
To verify NeuA has the ability to bind to FelD and prevent IgE engagement, We used blocking ELISA to test the blocking activity of different proteins. Compared with the control groups, absorbance at 450 nm of NeuB has an obvious decrease. Additionally, the combination of NeuA and NeuB have better blocking effect (Figure 5). The results of our experiment showed that NeuB has a blocking effect on antigens, and it has a better blocking effect when used together with NeuA. Due to the limited number of sera from allergic donors, we could not have sufficient materials for pre-experiments to reduce absorbance, but the high difference was enough to indicate that our scFvs have blocking activity.
Reference
[1] Orengo JM, Radin AR, Kamat V, Badithe A, Ben LH, Bennett BL, Zhong S, Birchard D, Limnander A, Rafique A, Bautista J, Kostic A, Newell D, Duan X, Franklin MC, Olson W, Huang T, Gandhi NA, Lipsich L, Stahl N, Papadopoulos NJ, Murphy AJ, Yancopoulos GD. Treating cat allergy with monoclonal IgG antibodies that bind allergen and prevent IgE engagement. Nat Commun. 2018 Apr 12;9(1):1421.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 679
Illegal SpeI site found at 75 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 679
Illegal SpeI site found at 75 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 679
Illegal XhoI site found at 276 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 679
Illegal SpeI site found at 75 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 679
Illegal SpeI site found at 75 - 1000COMPATIBLE WITH RFC[1000]