Expression and characterization of Gag protein of HIV-1(CN) in Pichia pastoris.

To express the core protein of HIV-1 of Chinese prevalent strain (HIV-1(CN)) in Pichia pastoris, the full-length gag gene was inserted into the secretory expression vector pHILS1. Linearized recombinant plasmid pHILGAG by SalI was electrotransformed into the yeast strain GS115, and the yeast transformants were identified by PCR. To induce the interest protein to be expressed, the PCR positive transformants were inoculated in the medium of BMGY and BMMY, mRNA of the strain was detected by RT-PCR, and the expressed protein was analyzed by SDS-PAGE, Western blotting and thin layer scanning. mRNA (1.3kb) was amplified by RT-PCR. SDS-PAGE and Western blotting analysis showed that the molecular mass of the expressed protein was 55kDa, which was similar to the expected value, and the expressed protein could react with McAb to HIV-1 p24. Thin layer scanning analysis demonstrated that the whole amount of the expressed protein was approximately 13% of the soluble protein in the supernatant. The recombinant yeast had good genetic stability. The optimal expression conditions of the engineering yeast were as follows: BMMY medium, 80-90% of dissolved oxygen, 1% methanol, and 3-day-cultivation course. Gag proteins were expressed under the optimal expression condition and purified via gel filtration chromatography. The purity of the interest protein was up to 85%. After the purified proteins were inoculated into BALB/c mice, the anti-HIV-1 antibodies in the immunized mice could be detected by Western blotting.

Purification and reactiongenicity of human immunodeficiency virus type 2 total external glycoprotein expressed in Pichia Pastoris.

The purification of human immunodeficiency virus type 2 total external glycoprotein gp105 expressed in Pichia pastoris was investigated. Expression conditions were optimized by an orthogonal test. The results from tests of variance analyses showed that the most important parameter for efficient expression of total gp105 in P. pastoris is adequate aeration during methanol induction. The optimum induction conditions for gp105 expression were: more than 85% aeration, induction for 3 days, the initial pH 6.0-7.0 and a final methanol concentration of 1.0%. Under these conditions, the expressed total gp105 was secreted into fermentation broth and reached a yield of 23%, approximately 141 mg/l. Expressed gp105 was isolated and purified by salting out and Sephadex G-100 chromatography and the yield of gp105 was 40%. gp105 was purified to electrophoretic purity and its isoelectric point (pI) was about 5.2 by SDS-PAGE and isoelectrofocusing. The purified gp105 contained approximately 35% carbohydrate, which proved that the expressed gp105 was a glycoprotein. Its N-terminal amino acid was arginine by Dansyl-Cl and the result indicated that expressed gp105 was secreted and cleaved correctly. The results from gp105 ELISA demonstrated that the purified total gp105 showed good reactiongenicity and antigenic specificity.

Academic achievement despite child maltreatment: a longitudinal study.

PURPOSE: Although researchers have concluded that child maltreatment has a negative effect on children's learning and academic achievement, not all children are negatively affected by maltreatment, and some children seem to succeed academically despite being maltreated. Drawing on risk and resilience theory, we examined a broad range of potential risk, promotive, and protective factors within children and their environments along with characteristics of the maltreatment to account for variability in test scores. METHODS: A national longitudinal probability sample of 702 maltreated school-aged children, ages 6-10, and their caregivers was used to predict reading and math scores among maltreated children over three years. RESULTS: We found that chronic maltreatment, poorer daily living skills, and lower intelligence explained a substantial proportion of the variance in maltreated children's math scores (39%), whereas type of maltreatment, poorer daily living skills and lower intelligence explained a substantial proportion of the variance in reading scores (54%) over time. Contrary to our prediction, having a behavior problem seemed to protect chronically maltreated children from poorer performance in math over time. CONCLUSIONS: To increase academic achievement among maltreated children, it is imperative that we prevent chronic maltreatment and help children increase their competency on daily living skills.