To develop an improved baculovirus insecticide with additional advantages, a novel recombinant baculovirus, AcB5B-AaIT was constructed. B. thuringiensis crystal protein gene (cry1-5) and insect-specific neurotoxin gene (AaIT) were introduced into Autographa californica nucleopolyhedrovirus genome by fusion of polyhedrin-cry1-5-polyhedrin under the control of polyhedrin (polh) gene promoter, and AaIT under the control of early promoter of ORF3004 from Cotesia plutellae bracovirus, respectively. About 150 kDa of Polyhedrin-Cry1-5-Polyhedrin fusion protein expressed by AcB5B-AaIT was occluded into the polyhedra produced by the recombinant virus, and activated as about 65 kDa of crystal protein when treated with gut-juice of Bombyx mori. The AcB5B-AaIT showed about 50% reduced LT50 value compared to that of the recombinant virus, Ap1Ac, expressing Cry1Ac against Plutella xylostella larvae. In addition, Spodoptera exigua larvae fed the recombinant polyhedra of AcB5B-AaIT showed about 4 fold higher refusing diet effect compared S. exigua larvae fed the recombinant polyhedra of the recombinant virus, Ap1C, expressing Cry1C. AcB5B-AaIT could be transferred to wild-type baculovirus along with serial passage by the homologous recombination between two polyhedrin genes contained in polh-cry1-5-polh fusion protein gene. These results suggested that the novel recombinant baculovirus, AcB5B-AaIT, could be applied as advanced viral insecticide.

This study was conducted to examine the effect of fermented alcoholic feedstuff (FAF), sustained-release recombinant bovine somatotropin(rBST) on blood metabolites and carcass characteristics of Korean native cattle, Hanwoo. The experiment was carried at the Livestock Breeding Station in Kangwon-do with fourteen bulls with two groups, control and treatment. Seven bulls per each group were allocated. In control group, bulls were treated with total mixed ration(TMR) for the whole experimental periods. In treatment group, bulls were treated whith TMR+FAF+rBST for the fattening period and TMR+FAF for finishing period, respectively. The results are summarized as follows ; Blood creatinine concentration was higher in treatment group than in control group(P<0.05), while cholesterol content in blood was lower in treatment group than in control group. There were no significant differences between in control and treatment group on back-fat thickness(BFT) and rib eye area(REA), however, MS of treatment was more improved than that of control. The frequencies of yield grade A and quality grade 1 were 33 and 17% for control group and, 50 and 50% for treatment group, respectively, which imply higher appearances of grade A and grade 1 in treatment group than in control group. The auction prices(won/carcass kg) were 11,145 and 11,573 won in control and treatment group, respectively, and net gross incomes per carcass were 654,408 and 1,051,542 won in control and treatment groups, respectively, which suggest that the margin of profit between treatment and control group was 397,134 won per carcass. Overall results indicate that the favorable feeding system to produce high quality beef with high profit is TMR+FAF+rBST feeding during the fattening period and TMR+FAF feeding during the finishing period.

This study was conducted to examine the effect of fermented alcoholic feedstuff (FAF), sustained-release recombinant bovine somatotropin(rBST) on growth performance and body size of Korean native cattle, Hanwoo. The experiment was carried at the Livestock Breeding Station in Kangwon-do with fourteen bulls of two groups, control and treatment. Seven bulls per each group were allocateed. In control group, bulls were treated with total mixed ration(TMR) for the whole experimental periods. In treatment group, bulls were treated with TMR+FAF+rBST for the fattening period and TMR+FAF for the finishing period, respectively. The results are summarized as follows ; Average daily gains(ADG) of control and treatment groups were 0.86 and 1.11kg during the fattening period. ADG of treatment group were 29.1% higher than that of control group. For the whole experimental period, the fattening period and finishing period, ADG in control and treatment groups were 0.69 and 0.79kg, respectively, which shows 14.5% improvement of ADG in treatment group. Dry matter intake(DMI) was higher in treatment group than in control group during the finishing, fattening or whole experimental period. Feed conversion efficiencies in fattening period were 8.65 for control group and 6.93 for treatment group which shows improving 19.9% of feed conversion efficiency for treatment group, while 12.38 for control group and 14.48 for treatment group in finishing period. There were no significant differences between control and treatment group in feed conversion efficiency for the whole experimental period. Overall results indicate that the favorable feeding system to produce growth performance is TMR+FAF+rBST feeding during the fattening period and TMR+FAF feeding in treatment during the finishing period.

This study was performed to examine the effects of recombinant bovine somatotropin (rBST) and fermented alcoholic feedstuff(FAF) on blood metabolites, carcass characteristics and profitability. Sixteen Hanwoos(bulls; 8, steers; 8) with average 143㎏ of body weight were randomly assigned to bull control group(BC), steer control group(SC), bull group(BrA) treated with rBST and FAF, and steer group(SrA) treated with rBST and FAF. Sustained release rBST(0.03㎎/㎏/body weight) was administered biweekly and dose of rBST was adjusted monthly according to body weight increase. The blood glucose content was higher in the groups treated with rBST and FAF than control groups. The blood cholesterol contents tended to be lower in the treatment groups (BrA, 120.5 mg/dl; SrA, 104.0 mg/dl) than in the control groups (BC, 141.0 mg/dl; SC, 125.5 mg/dl). No significant difference between the groups of bulls and steers were found in the blood triglyceride concentrations as well as the albumin contents. The rib eye area of BC and BrA were 70 and 72 cm2, respectively, which are slightly larger than the area of SC and SrA. The treatment did not affect the backfat thickness both in bulls and steers. The marbling scores of BC and BrA appeared as No. 1 and No. 2, respectively, and No. 3 and No. 4 were obtained in SC and SrA. Lower non saturate fat concentrations were observed in the treatment groups (BrA, 64.30%; SrA, 59.59) than in control groups (BC, 58.98%; SC, 58.09%). Saturated fatty acids and amino acids contents were slightly more improved in the treatment than in the control. Average auction prices of BC and BrA were 7,900 and 8,089 wons per 1kg of carcass, respectively, whereas the prices of SC and SrA were 9407 and 9866 wons per 1kg of carcass, respectively. It is concluded that the treatment with rBST and FAF can improve the growth performance such as marbling scores and rib eye area and increase the auction prices.

본 연구에서는 외래 유전자의 지속적인 발현에 의한 형질 전환 개체나 세포의 생리적인 부작용을 최소화하기 위하여 hTPO 유전자의 발현을 조절할 수 있는 tetracycline-inducible retrovirus vector system을 구축하고자 하였다. hTPO 유전자는 사람의 간암세포인 HepG2에서 분리한 RNA를 주형으로 하여 RT-PCR 방법을 이용하여 확보하였으며, 이 유전자를 MLV 유래의 vector에 도입하여 pLNChTPOW를 재조합하였다. 재조합한 vector는 GP2 293 포장세포에 도입하여 바이러스를 생산하였으며, 이 바이러스를 이용하여 감염시킨 여러 표적세포에서 hTPO의 발현을 확인하였다. 또한, hTPO의 발현을 유도적으로 조절할 수 있도록 하기 위하여 hTPO를 one vector 형태의 Tet-On vector system에 도입하였으며, 발현의 유도 조건에서 보다 강한 발현을 위하여 WPRE 서열을 여러 위치에 도입하였다. 구축한 Tet system의 발현 조절 정도는 각 바이러스를 감염시켜서 구축한 CEF와 PFF 세포에서 RT-PCR과 Western blot, 그리고 ELISA 방법을 이용하여 확인하였다. 그 결과, CEF에서는 WPRE 서열이 hTPO 유전자의 3'에 위치한 경우에서, PFF에서는 WPRE가 rtTA의 3'에 위치한 경우의 vector system에서 가장 높은 발현율과 유도율을 나타내었다. 이는 Tet system에서의 hTPO 유전자 발현 조절이 매우 효율적으로 이루어지며, 세포주에 따른 의존적인 조절 양상을 보이는 것을 의미한다. 따라서 hTPO의 대량 생산을 위한 생체 반응기로서의 형질 전환 동물의 개발을 보다 효율적으로 수행하려면 적절한 Tet system이 선별적으로 적용되어야 할 것이다.

This study was conducted to examine the effect of IRES controlled reporter gene on screening and production of recombinant human erythropoietin (EPO) proteins from cultured CHO cells. The cDNA was cloned for EPO from human liver cDNA. Using site-directed mutagenesis, we generated recombinant human EPO (rhEPO) with two additional N-glycosylations (Novel erythropoiesis-stimulating protein: NESP). Wild type hEPO and NESP were cloned into expression vectors with GFP reporter gene under regulatory control of CMV promoter and IRES so that the vectors could express both rhEPO and GFP. The expression vectors were transfected to cultured CHO-K1 cells. Under microscopy, expression of GFP was visible. Using supernatant of the culture, ELISA assay, immunocytochemistry and in vitro assay using EPO dependant cell line were performed to estimate biological activity to compare the production characteristics (secretion levels, etc.) between rhEPO and NESP. The activity of NESP protein, obtained by mutagenesis, was described and compared with its rhEPO counterpart produced under same conditions. Although NESP had less secretion level in CHO cell line, the biological activity of NESP was greater than that of rhEPO. These results are consistent with previous researches. We also demonstrated that rhEPO and GFP proteins expressed simultaneously from transfected CHO cell line. Therefore we conclude that use of GFP reporter gene under IRES control could be used to screen and produce rhEPO in cultured CHO cells.

The hematopoietic growth factor erythropoietin (EPO) is required for the maintenance, proliferation, and differentiation of the stem cells that produce erythrocytes. To analyse the biological activity of the recombinant human EPO (rec-hEPO), we have cloned the EPO cDNA and genomic DNA and produced rec-hEPO in the CHO cell lines. The growth and differentiation of EPO-dependent human leukemic cell line (F36E) were used to measure cytokine dependency and in vitro bioactivity of rec-hEPO. MIT assay values were increased by survival of F36E cells at 24h or 72h. The hematocrit and RBC values were increased by subcutaneous injection of 20 IU (in mice) and 100IU(in rats) rec-hEPO. Hematocrit values remarkably increased at 13.2% (in mice) and 12.2% (in rats). The pharmacokinetic behavior with injection of 6 IU of rec-hEPO remained detectable after 24 h in all mice tested. The highest peat appeared at 2h after injection. The long half-life of rec-hEPO is likely to confer clinical advantages by allowing less frequent dosing in patients treated for anemia. These data demonstratethat ree-hEPO produced in this study has a potent activity in vivo and in vitro. The results also suggest that biological activity of ree-hEPO could be remarkably enhanced by genetic engineering that affects the potential activity, including mutants with added oligosaccharide chain and designed to produce EPO-EPO fusion protein.