Expression Study of a Recombinant Plasmid containing Dipeptidyl Peptidase-4 Gene in E. coli: A Plausible Application for Celiac Disease Patients to Digest Gluten원문보기
Lee, Yeonjae
(Hankuk Academy of Foreign Studies)
,
Kang, Ryan
(Hankuk Academy of Foreign Studies)
,
Kwon, Jenna
(Hankuk Academy of Foreign Studies)
,
Jo, Kyuhee
(Hankuk Academy of Foreign Studies)
,
Im, Jungbin
(Hankuk Academy of Foreign Studies)
,
Jung, Sangwook
(Hankuk Academy of Foreign Studies)
,
Lee, DongHyun
(Hankuk Academy of Foreign Studies)
,
Lee, Juhyeon
(Hankuk Academy of Foreign Studies)
,
Lee, Jeong-Sang
(Dep. of Biotechnology and Functional Foods, Jeonju University)
Celiac disease (CD) is an immune-mediated enteropathy of small intestine diagnosed in both childhood and adulthood. CD is caused by gluten, which produces gliadorphin during its digestion. The enzyme dipeptidyl peptidase-4 (DPP4) breaks gliadorphin down nevertheless the last tripeptide remains and e...
Celiac disease (CD) is an immune-mediated enteropathy of small intestine diagnosed in both childhood and adulthood. CD is caused by gluten, which produces gliadorphin during its digestion. The enzyme dipeptidyl peptidase-4 (DPP4) breaks gliadorphin down nevertheless the last tripeptide remains and eventually inhibits DPP4, thus slowing down its process. Therefore, the idea is to produce an additional DPP4 enzyme which is crucial. Consequently, the functional DPP4 gene was cloned into pCDNA3 intermediate (FLAG+DPP4) vector and finally a recombinant plasmid pSB1C3 (Andersons promoters+FLAG+DPP4) was constructed using synthetic biology. Normally, a DPP4 inhibitor is used as a cure for diabetes. Another important concern was overexpression of DPP4, which might lead to diabetes, accordingly the work was also performed for the regulation of the DPP4 gene expression. In this regard, three types of Anderson promoters (strong, moderate and weak) were utilized to study the control overexpression. This is the first report of idealistic trial for control the exogenous DPP4 gene-expression by molecular biologic tools.
Celiac disease (CD) is an immune-mediated enteropathy of small intestine diagnosed in both childhood and adulthood. CD is caused by gluten, which produces gliadorphin during its digestion. The enzyme dipeptidyl peptidase-4 (DPP4) breaks gliadorphin down nevertheless the last tripeptide remains and eventually inhibits DPP4, thus slowing down its process. Therefore, the idea is to produce an additional DPP4 enzyme which is crucial. Consequently, the functional DPP4 gene was cloned into pCDNA3 intermediate (FLAG+DPP4) vector and finally a recombinant plasmid pSB1C3 (Andersons promoters+FLAG+DPP4) was constructed using synthetic biology. Normally, a DPP4 inhibitor is used as a cure for diabetes. Another important concern was overexpression of DPP4, which might lead to diabetes, accordingly the work was also performed for the regulation of the DPP4 gene expression. In this regard, three types of Anderson promoters (strong, moderate and weak) were utilized to study the control overexpression. This is the first report of idealistic trial for control the exogenous DPP4 gene-expression by molecular biologic tools.
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문제 정의
To our knowledge, this is the first report of idealistic trial for control the exogenous production of DPP4 using synthetic biology. In the future, after the synthesis of the recombinant plasmid and checking the relevant parameters, TNCR Korea will be designing the new gut bacteria and will use further in probiotic yogurts to be safely consumed by gluten intolerance patients.
제안 방법
After confirmation of recombinant plasmid, the next task was to create the backbone plasmid by the final addition of insert gene(anderson+FLAG+DPP4) into pSB1C3. The desired plasmid was created by insertion of Anderson promoter in the backbone through PCR and ligation to the final vector (pSB1C3).
The DPP4 thermocycling was performed in triplicate using an automated temperature-control system (TaKaRa Korea Biomedical Inc., Seoul) using primers 5′-TGA CGA CAA GGG ATC CAT GAA GAC ACC GTG-3′ and 5′-TAG ATG CAT GCT CGA GCT AGT GTA AGG AGA AGC ACT-3′ as forward and reverse primers (as described above), respectively PCR reaction mixture (50 µl) consisted of 1 µl of pCDNA3 DNA (100 ng), 2 µl of each (forward and reverse) primer (10 pmoles/ µl), 4 µl of dNTP mix, 1 µl of GXL DNA polymerase, 10µl of GXL PCR buffer and 30 µl of distilled water.
대상 데이터
As per the requirement of the project, DPP4 gene was amplified from vector pCMV SPORT6 kindly provided from Korea Human Gene Bank, Medical Genomics Research center, KRIBB, Korea. The primers for the amplification of DPP4 gene were designed and consisted of vector sequence (pCDNA), restriction enzyme sequence (RE) and DPP4 gene sequence (Fig.
성능/효과
In conclusion, we successfully developed a modified plasmid using synthetic biology with the transformation of E. coli, which secretes DPP4 with optimized promoter conditions. Importantly, we clearly demonstrated that infusion cloning worked for the construction of the desired vector and confirmed the expression of the protein.
The results of the PCR amplification and agarose gel electrophoresis showed the presence of gene fragment bands of 2340 bp, which confirmed that the DPP4 gene was successfully integrated into the genome of E. coli (Anderson-33 bp+Flag-18 bp + EcoR1-6 bp +DPP4-2283 bp) (Fig. 5). After purification, the 87 ng/µl, 92 ng/µl and 94 ng/µl concentrations of the DNA were used for amplification with primers consisted of A1, A2, and A3 respectively.
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