• 생명과학회지
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• 제11권4호
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• pp.385-391
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• 2001

Filamentous hemagglutinin (FHA) is considered as an essential immunogenic component for incorporation into acellular vaccines against Bordetella pertussis, the causative agent of whooping cough. Classically, antipertussis vaccination has employed an intramuscular route. An alternative approach to stimulate mucosal and systemic immune responses is oral immunization with recombinant live vaccine carrier strains of Salmonella typhimurium. An attenuated live Salmonella vaccine sgrain($\Delta$cya $\Delta$crp) expressing recombinant FHA(rFHA) was developed. Stable expressionof rFHA was achieved by the use of balanced-lethal vector-host system. which employs an asd deletion in the host chromosome to impose in obligate requirement for diaminopimelic acid. The chromosomal $\Delta$asd mutation was complemented by a plasmid vector possessing the asd$^{+}$ gene. A 3 kb DNA fragment encoding immuno dominant regionof FHA was subcloned in-frame downstream to the ATG translation initiation codon in the multicopy Asd$^{+}$ pYA3341 vector to create pYA3457. Salmonella vaccine harboring pYA3457 expressed approximately 105kDa rFHA protein. The 100% maintenance of [YA3457 in vaccine strain was confirmed by stability examinations. Additionally, a recombinant plasmid pYA3458 was constructed to overpress His(8X)-tagged rFHA in Essherichia coli. His-tagged rFHA was purified from the E. coli strain harboring pYA3458 using Ni$^{2+}$-NTA affinity purification system.>$^{2+}$-NTA affinity purification system.

• BMB Reports
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• 제39권1호
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• pp.22-25
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• 2006

The gene encoding MPB83 from Mycobacterium bovis Vallee111 chromosomal DNA was amplified by using polymerase chain reaction (PCR) technique, and the PCR product was approximately 600bp DNA segment. Using T-A cloning technique, the PCR product was cloned into pGEM-T vector and the cloning plasmid pGEM-T-83 was constructed successfully. pGEM-T-83 and pET28a(+) were digested by BamHI and EcoRI double enzymes. The purified MPB83 gene was subcloned into the expression vector pET28a(+), and the prokaryotic expression vector pET28a-83 was constructed. Plasmid containing pET28a-83 was transformed into competence Escherichia coli BL21 (DE3). The bacterium was induced by isopropyl-$\beta$-D-thiogalactopyranoside (IPTG) and its lysates were loaded directly onto sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), approximately 26 kDa exogenous protein was observed on the SDS-PAGE. The protein was analyzed using Western-blotting. The results indicated that the protein was of antigenic activity of M. bovis. The results were expected to lay foundation for further studies on the subunit vaccine and DNA vaccine of MPB83 gene in their prevention against bovine tuberculosis.

• Journal of Microbiology and Biotechnology
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• 제2권4호
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• pp.237-242
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• 1992

In order to clone the gene coding for alkaline phosphatase in the yeast Kluyveromyces fragilis, a genomic library was constructed using the yeast-E. coli shuttle vector pHN114 as a cloning vector. From the genomic library, a clone carrying the gene was isolated and the plasmid was designated as pSKH101. A restriction enzyme map was made using this plasmid. Subcloning experiments and complementation studies showed that alkaline phosphatase was active only in the original 3.1 kb insert. Southern hybridization analysis confirmed that the cloned DNA fragment was derived from K. fragilis genomic DNA. Using a minicell experiment, the product of the cloned gene was identified as a protein with a molecular weight of 63 KDa. A 0.6 kb HindIII fragment, which showed promoter activity, was isolated using the E. coli promoter-probe vector pKO-1.

• Journal of Microbiology and Biotechnology
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• 제9권2호
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• pp.230-233
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• 1999

A plasmid vector was constructed for the expression and secretion of Bacillus macerans cyclodextrin glucanotransferase (CGTase) in Bacillus subtilis. The vector, pUBACGT, was composed of the ribosome-binding sequence, signal sequence, and cgt gene from B. macerans under the control of amyR2, the promoter of amyE gene coding for $\alpha$-amylase from B. subtilis var. natto. Bacillus subtilis LKS88, a mutant strain lacking genes for an amylase and two proteases, was used as a host for the transformation of the plasmid vector. The transformants were selected on kanamycin-containing Luria-Bertani plates. The starch hydrolyzing activity was observed on the starch-containing plates by the iodine method and cyclodextrin-forming activity was detected in the culture medium. A SDS-PAGE analysis showed that most of the expressed CGTase in the recombinant B. subtilis was secreted into the medium at a high expression level.

• 한국미생물·생명공학회지
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• 제18권3호
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• pp.305-308
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• 1990

Shuttle plasmid vector인 pHN114를 이용하여 Kluyveromyces fragilis의 3-isopropylmalate dehydrogenase 유전자를 cloning 하였다. 그 결과 Saccharomyces cerevisiae의 leu2변이와 E.coli의 leuB변이를 상보하는 두가지 clone 체 pJK104와 pJK106을 얻었다. Restriction mapping 결과 이들은 서로 반대방향으로 삽입되어 있었으며 expression activity는 pJK104가 높았다. pJK104에 삽입된 유전자를 BglII와 SalI으로 끊은 1.6kb fragment를 probe 로 하여 Southern Hybridization 한 결과 유전자의 유래가 Kluyveromyces fragilis 임을 확인하였다.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2004년도 Annual Meeting BioExibition International Symposium
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• pp.323-327
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• 2004

Leuconostoc citreum CBUE isolated from kimchi proved to harbor a small cryptic plasmid, pNS75. The complete nucleotide sequence of pNS75 was 1,821 bp and had a low G+C content of 39.2%. Computer analysis using DNASIS revealed one open reading frame (ORF), having ATG as putatitive start condon and potentially encoding proteins with molecular mass of 38 kDa. The chimeric plasmid pLeuCM was first constructed wih pNS75, pUC19 and chroamphenicol acetyltransferase (CAT) from Staphylococcus sp.. pLeuCM replicated and expressed chroamphenicol acetyltransferase in Leuconostoc citerum CBNF after transformation. To test the availability of shuttle vector as cloning vehicle of foreign gene, $\alpha$-amylase gene of Streptococcus bovis was cloned and all transformants secreated the $\alpha$-amylase successfully. The result indicates that pLeuCM is a potential shuttle vector for Leuconostoc spp. and lactic acid bacteria.

• 한국축산식품학회지
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• 제39권1호
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• pp.13-22
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• 2019

This study aimed to develop a bile-responsive expression system for lactobacilli. The promoters of four genes, encoding phosphoenolpyruvate-dependent sugar phosphotransferase (mannose-specific), L-lactate dehydrogenase (LDH), HPr kinase, and D-alanine-D-alanine ligase, respectively, which were highly expressed by bile addition in Lactobacillus johnsonii PF01, were chosen. Each promoter was amplified by polymerase chain reaction and fused upstream of the ${\beta}$-glucuronidase gene as a reporter, respectively. Then, these constructs were cloned into E. coli-Lactobacillus shuttle vector pULP2, which was generated by the fusion of pUC19 with the L. plantarum plasmid pLP27. Finally, the constructed vectors were introduced into L. plantarum for a promoter activity assay. The LDH promoter showed the highest activity and its activity increased 1.8-fold by bile addition. The constructed vector maintained in L. plantarum until 80 generations without selection pressure. A bile-responsive expression vector, $pULP3-P_{LDH}$, for Lactobacillus spp. can be an effective tool for the bile-inducible expression of bioactive proteins in intestine after intake in the form of fermented dairy foods.

• Parasites, Hosts and Diseases
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• 제60권3호
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• pp.201-205
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• 2022

Babesia microti is one of the most common causative agents of babesiosis. A sensitive and rapid detection is necessary for screening potentially infected individuals. In this study, B. microti cytochrome c oxidase subunit I (cox1) was selected as the target gene, multiple primers were designed, and optimized by a recombinase-aided amplification (RAA) assay. The optimal primers and probe were labeled with fluorescein. The sensitivity of fluorescent RAA (fRAA) was evaluated using gradient diluents of the cox1 recombinant plasmid and genomic DNA extracted from whole blood of B. microti infected mice. The specificity of fRAA was assessed by other transfusion transmitted parasites. The analytical sensitivity of the fRAA assay was 10 copies of recombinant plasmid per reaction and 10 fg/µl B. microti genomic DNA. No cross-reaction with any other blood-transmitted parasites was observed. Our results demonstrated that the fRAA assay would be rapid, sensitive, and specific for the detection of B. microti.

• 한국미생물·생명공학회지
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• 제15권6호
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• pp.396-401
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• 1987

$\beta$-Galactosidase 유전자 조작된 두 대장균 숙주-벡터 시스템의 단백질 생산가 배양특성을 비교하였다. Tac 프로모터를 갖는 Escherichia coli JM109/ pTBG10 균주가 pL 프로모터를 갖는 E. coli MH 3000(pRKcI857)/ pASl(lacz) 균주보다도 단백질 생산성에 있어서 훨씬 우수하였으며 초기 및 중간 대수 증식기에서의 발현 유도가 단백질 생산에 있어서 유리함을 관찰하였다. 또한 유전자 발현시기에 있어서는 산소요구량이 매우 높았으나 pH를 일정하게 조절함에 의해 산소요구량을 어느 정도 낮출 수 있었고 생산성도 향상됨을 관찰할 수 있었다. 고생산성 균주(E. coli JM109/pTBG10)의 경우 플라스미드 함유 균체의 플라스미드 상실 균체에 대한 비증식속도의 비 $\mu$ + /$\mu$- 가 저생산성 균주보다도 낮았으며 이것은 유전자발현에 따른 단백질 합성부담이 매우 큰 때문으로 판단되었다. 또 플라스미드 안정성을 30세대까지 추적하였는바 유전자 발현 조건하에서 두 균주 모두 25세대 후 10% 정도의 안정성을 보였으나 E. coli JM109/pTBG10 균주의 안정성이 상대적으로 우수하다고 판단되었다.

• 미생물학회지
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• 제28권1호
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• pp.41-46
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• 1990

자연환경으로부터 분리한 DJ-12 균주는 4CBA 및 4CB를 비롯하여 그 대사산물인 4OHBA와 PCA를 분해하여 단일 탄소원으로 이용하였다. DJ-12 균주에서 4CBA 및 4CB분해유전자는 약 65kb 크기의 plasmid인 pDJ121에 존재하였으며, 이 pDJ121은 ExoRI, HindIII, SalI 그리고 PslI의 절단부위를 각각 9, 11, 10 그리고 19개씩 가지고 있었다. EcoRI으로 처리한 pDJ121 절편을 pKT230에 ligation 시켜 재조합 vector인 pDK450을 만들었으며, 이를 Pseudomonas putida KT2440에 transformation 시켜 얻은 cloned cell 에서는 4CBA 분해유전자가 잘 발현되었다.