• 대한수의학회지
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• 제44권4호
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• pp.625-635
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• 2004

Trichnuris suis does not excrete eggs during larval stage as well as in particular adult stage, It is impossible to diagnose by use of fecal examination method in those periods. Therefore, serological diagnostic method can be very useful for those stages. In order to produce monoclonal antibody, specific somatic and secretory-excretory (SE) antigens of T. suis were identified and analyzed by SDS-PAGE and Western blot. Monoclonal antibody-producing hybridoma cells were cloned, which were made of popliteal lymph node of BALB/c mice immunized with a 45 kDa somatic antigen of T. suis. Five clones (1B9, 2C4, n2C5, 2D7 and 2D8) showing strong responses to T. suis antigens were selected and the isotype identified. All monoclonal antibodies were IgG1 isotype and the light chains were k chain. Established monoclonal antibodies reacted specifically to somatic and SE antigens of T. suis and did not cross-reacted to antigens of ascaris suum, trichuris vulpis, or Trichinella spiralis. The sensitivity of somatic and SE antigens against these monoclonal antibodies were significant (p<0.01) associated with those of positive and negative sera.

• Asian-Australasian Journal of Animal Sciences
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• 제13권4호
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• pp.440-445
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• 2000

Thirty lactating Javanese thin-tail ewes (12 ewes had been injected, prior to mating, with 700 IU pregnant mare serum gonadotropin, and 18 ewes with saline as a control) were used to evaluate the effect of superovulation on milk production during lactation and mammary chemical indices at the end of lactation. Thirteen ewes (9 control and 4 superovulated ewes) were fed at low and the other 17 ewes (9 control and 8 superovulated ewes) were fed at high quality ration. Superovulated ewes, either fed at low or high quality ration, had dramatically higher milk yields (57%). At the end of lactation, superovulated ewes had higher mammary dry fat-free tissue, mammary DNA concentration, total mammary DNA and RNA contents than nonsuperovulated ewes. Superovulation did not affect mammary RNA and collagen concentrations, and total collagen content. Ration quality did not significantly increase milk production during lactation and mammary chemical indices at the end of lactation. The observed increase in milk production in the superovulated ewes was probably due to the increased mammary secretory cell number and their synthetic activities during lactation as a result of the increased endogenous hormonal stimulation of mammary growth and development during pregnancy.

• Journal of Microbiology and Biotechnology
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• 제23권8호
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• pp.1116-1122
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• 2013

Cell line development is the most critical and also the most time-consuming step in the production of recombinant therapeutic proteins. In this regard, a variety of vector and cell engineering strategies have been developed for generating high-producing mammalian cells; however, the cell line engineering approach seems to show various results on different recombinant protein producer cells. In order to improve the secretory capacity of a recombinant tissue plasminogen activator (t-PA)-producing Chinese hamster ovary (CHO) cell line, we developed cell line engineering approaches based on the ceramide transfer protein (CERT) and X-box binding protein 1 (XBP1) genes. For this purpose, CERT S132A, a mutant form of CERT that is resistant to phosphorylation, and XBP1s were overexpressed in a recombinant t-PA-producing CHO cell line. Overexpression of CERT S132A increased the specific productivity of t-PA-producing CHO cells up to 35%. In contrast, the heterologous expression of XBP1s did not affect the t-PA expression rate. Our results suggest that CERT-S132A-based secretion engineering could be an effective strategy for enhancing recombinant t-PA production in CHO cells.

• 대한수의학회지
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• 제40권1호
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• pp.130-137
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• 2000

In order to diagnose canine heartworm infection by antigen capture ELISA, the crude somatic(S), partial somatic(below 45kDa) and excretory/secretory(E/S) antigen of adult heartworm were identified and the antigenicity was examined by silver stain, immunoblot and ELISA. Then, production of monoclonal antibody to specific antigen carried out in this experiment. The bands to S antigen and E/S antigen were recognized between 10 and 200kDa and common bands were recognized strongly 14, 18, 28, 43kDa by silver stain. By western blot analysis, fractions to S antigen were recognized 14, 16, 18, 20, 24, 28, 32, 43, 50, 55kDa, etc. and only a 14kDa to E/S antigen in positive sera which were positive in modified Knott's test and necropsy. In ELISA, the positive sera reacted to antigens(SA, $SA_{45}$, E/S) were significantly different from negative sera by Student's t-test(p<0.05). Four hybridoma cell lines(14, 16, 17, 32kDa) than produce specific monoclonal antibodies for these antigens were obtained by immunizing BALB/c mice with a partially purified somatic antigen (below 45kDa) preparation, by fusing spleen cells with SP2/O cell myeloma cells, and by screening cell culture supernatants for antibody. In these results, it was confirmed that partial somatic antigen(below 45kDa) or E/S antigen can be used for serologic diagnosis of heartworm infection and monoclonal antibody reacting with specific antigen(14kDa) can be used for antigen capture ELISA in prepatent period of canine heartworm infection.

• 생명과학회지
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• 제17권6호통권86호
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• pp.847-858
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• 2007

ER-Golgi 분비 경로를 통해서 정확한 구조를 가지면서 post-translational modification 과정을 거친 재조합 단백질의 발현을 최대화하는 것은 ER stress반응에 대한 연구의 중요한 계기가 된다. 세포가 스트레스를 받지 않는 상태라도 ER stress signaling은 재조합 단백질의 생산량을 제한하고 품질을 떨어뜨리는 여러 가지 조건을 만들게 된다. ER stress signaling을 막는 여러 가지 방법들이 제시되고 있으며 표 2는 이러한 방법들 중 일부를 나타내고 있다. 일반적으로는 pro-survival 경로에 관련되어 있는 인자를 촉진하고 pro-apoptosis에 관련되어 있는 인자를 억제하는 것들이다. 그러나 ER stress 반응은 매우 복잡하고 적응과 사멸 기작(adaptation and elimination mechanism)의 중간 역할을 하기 때문에 ER stress에 관련된 주요 인자를 산업적으로 응용하기 위해선 이들의 기능에 대해 보다 깊은 연구가 이루어져야 한다. 현재까지 재조합단백질의 생산량을 최대한으로 높이는 방법은 ER stress 반응이 생기지 않도록 fed-batch process를 개선하고 세포 사멸 기작을 조절하며 단백질의 glycosylation 처리를 하는 것이다.

• 생명과학회지
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• 제17권11호
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• pp.1601-1604
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• 2007

Agarivorans sp. JA-1 유래의 내열성 ${\beta}-agarase$ 유전자의 분비형 과발현과 재조합효소분해산물의 항균효과를 확인하였다. B. subtilis 168, DB104, ISW1214의 3가지 재조합발현 숙주를 비교하여 ISW1214 균주가 LB배지에서 배양 9시간에 총 효소활성 52,460 U과 비활성 201 U/mg의 최대 발현량을 보이는 것을 확인하였다. 이는 E. coli BL21 균주에 비해 총 효소활성은 약 90배, 비활성은 약 100배 증가한 결과이다. 재조합 ${\beta}-agarase$의 기질로 저가의 한천을 사용하여 neoagarobiose, neoagarotetraose 등 의 neoagarooligosaccharide를 생산하였으며, 생산된 neoagarooligosaccharides는 그람양성 세균인 B. subtilis와 그람음성 세균인 E. coli 모두의 성장을 저해하는 항균활성이 있음을 확인하였다. 따라서 본 연구에서 생산된 재조합 ${\beta}-agarase$와 재조합 효소의 한천분해 산물은 식품산업 등에 사용가능한 천연 항균제 생산에 활용이 가능할 것으로 기대된다.

• KSBB Journal
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• 제26권5호
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• pp.427-432
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• 2011

In this study, a EXGA gene code for exo-β-1,3-glucanase from Aspergillus oryzae was overexpressed and secretory produced in Saccharomyces cerevisiae. To overexpress the β-1,3-glucanase, pGInu-exgA and pAInu-exgA plasmids having GAL10 and ADH1 promoter, respectively, and exoinulinase signal sequence (Inu s.s) were constructed and introduced in S. cerevisiae SEY2102 and 2805. The recombinant β-1,3-glucanase was successfully expressed and secreted into the medium and the β--1,3-glucanase activity in 2102/pGInu-exgA and 2102/pAInu-exgA strain were 5.01 unit/mL and 4.09 unit/mL, respectively. In the 2805/pGInu-exgA and 2805/pAInu-exgA strain, the β-1,3-glucanase activity showed 3.23 unit/mL and 3.22 unit/mL, respectively. Secretory efficiency in each strain reached 95% to 98%. Subsequently, the recombinant β1,3-glucanase was used for ethanol production. Ethanol productivity in 2102/pAInu-exgA strain was 0.83 g/L when pre-treated Laminaria japonica which has initial reducing sugar of 1.4 g/L was used as substrate. It is assumed that the polysaccharides of Laminaria japonica was effectively saccharified by recombinant β-1,3-glucanase, resulting in increase of ethanol productivity. These results suggested that recombinant β-1,3-glucanase was efficiently overexpressed and secreted in S. cerevisiae SEY2102 as host strain by using ADH1 promoter-Inu s.s system.

• 생명과학회지
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• 제23권7호
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• pp.863-868
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• 2013

Bacillus sp. HY-20균주 유래 endoxylanase를 코드하는 XylP 유전자를 효모에서 발현시키기 위해 두 개의 발현 플라스미드 pG-xylP와 pGMF-xylP를 구축하였다. 이들 플라스미드는 endoxylanase의 분비발현을 위해 각각 다른 분비서열인 XylP 유전자의 자체 분비서열(XylP s.s)과 최적화된 $MF{\alpha}$ 분비서열($MF{\alpha}_{opt}$ s.s)을 가지고 있으며, S. cerevisiae SEY2102와 FY833균주에 형질전환되어 그 분비활성이 비교 조사되었다. 재조합 endoxylanase는 분비발현시스템과 숙주세포에 따라 23.7~70.1 unit/ml의 활성으로 효모 세포에서 성공적으로 발현되었고, 그 중 SEY2102/pGMF-xylP 형질전환주를 이용해 baffled-flask 배양을 실시한 결과 최대 88.1 unit/ml의 endoxylanase 활성을 보임을 확인하였다. 대부분의 재조합 endoxylanase는 세포 외 분획에 효율적으로 분비 생산되었으며, $MF{\alpha}_{opt}$ 분비서열이 XylP 유전자의 자체 분비서열보다 endoxylanase를 더 효율적으로 분비시킴을 확인할 수 있었다. 그러므로 본 연구에서 개발된 발현시스템은 효모를 숙주세포로 하여 많은 양의 세포 외 endoxylanase의 생산을 가능하게 하고, 바이오에탄올 생산 및 산업적 응용에도 유용하게 사용 될 수 있으리라 기대된다.

• Journal of Ginseng Research
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• 제35권2호
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• pp.176-190
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• 2011

There seems to be some controversy about the effect of total ginseng saponin (TGS) on the secretion of catecholamines (CA) from the adrenal gland. Therefore, the present study aimed to determine whether TGS can affect the CA release in the perfused model of the adrenal medulla isolated from spontaneously hypertensive rats (SHRs). TGS (15-150 ${\mu}g/mL$), perfused into an adrenal vein for 90 min, inhibited the CA secretory responses evoked by acetylcholine (ACh, 5.32 mM) and high $K^+$ (56 mM, a direct membrane depolarizer) in a dose- and time-dependent fashion. TGS (50 ${\mu}g/mL$) also time-dependently inhibited the CA secretion evoked by 1.1-dimethyl-4 -phenyl piperazinium iodide (DMPP; 100 ${\mu}M$, a selective neuronal nicotinic receptor agonist) and McN-A-343 (100 ${\mu}M$, a selective muscarinic M1 receptor agonist). TGS itself did not affect basal CA secretion (data not shown). Also, in the presence of TGS (50 ${\mu}g/mL$), the secretory responses of CA evoked by veratridine (a selective $Na^+$ channel activator (50 ${\mu}M$), Bay-K-8644 (an L-type dihydropyridine $Ca^{2+}$ channel activator, 10 ${\mu}M$), and cyclopiazonic acid (a cytoplasmic $Ca^{2+}$-ATPase inhibitor, 10 ${\mu}M$) were significantly reduced, respectively. Interestingly, in the simultaneous presence of TGS (50 ${\mu}g/mL$) and N${\omega}$-nitro-L-arginine methyl ester hydrochloride [an inhibitor of nitric oxide (NO) synthase, 30 ${\mu}M$], the inhibitory responses of TGS on the CA secretion evoked by ACh, high $K^+$, DMPP, McN-A-343, Bay-K-8644, cyclopiazonic acid, and veratridine were considerably recovered to the extent of the corresponding control secretion compared with the inhibitory effect of TGS-treatment alone. Practically, the level of NO released from adrenal medulla after the treatment of TGS (150 ${\mu}g/mL$) was greatly elevated compared to the corresponding basal released level. Taken together, these results demonstrate that TGS inhibits the CA secretory responses evoked by stimulation of cholinergic (both muscarinic and nicotinic) receptors as well as by direct membrane-depolarization from the isolated perfused adrenal medulla of the SHRs. It seems that this inhibitory effect of TGS is mediated by inhibiting both the influx of $Ca^{2+}$ and Na+ into the adrenomedullary chromaffin cells and also by suppressing the release of $Ca^{2+}$ from the cytoplasmic calcium store, at least partly through the increased NO production due to the activation of nitric oxide synthase, which is relevant to neuronal nicotinic receptor blockade, without the enhancement effect on the CA release. Based on these effects, it is also thought that there are some species differences in the adrenomedullary CA secretion between the rabbit and SHR.

• 한국미생물·생명공학회지
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• 제48권1호
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• pp.32-37
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• 2020

We improved on a unified saccharification and fermentation (USF) system for the direct production of ethanol from agarose by increasing total agarase activity. The pGMFα-NGH plasmid harboring the NABH558 gene encoding neoagarobiose hydrolase and the AGAG1 and AGAH71 genes encoding β-agarase was constructed and used to transform Saccharomyces cerevisiae 2805. NABH558 gene transcription level was increased and total agarase activity was increased by 25 to 40% by placing the NABH558 gene expression cassette upstream of the other gene expression cassettes. In the 2805/pGMFα-NGH transformant, three secretory agarases were produced that efficiently degraded agarose to galactose, 3,6-anhydro-L-galactose (AHG), neoagarobiose, and neoagarohexaose. During the united cultivation process, a maximum of 2.36 g/l ethanol from 10 g/l agarose was produced over 120 h.