ABSTRACT
<p><b>OBJECTIVES</b>Heparin-binding neurite-promoting factor (HBNF) is a heparin-binding protein primarily found in the brain, which can stimulate neurite outgrowth in vitro. We expressed recombinant human heparin-binding neurite-promoting factor (hrHBNF) using a yeast system, and observed its activity in stimulating neurite outgrowth in vitro.</p><p><b>METHODS</b>cDNA encoding mature human HBNF was amplified from total RNA isolated from an 18-week aborted human fetal brain by RT-PCR method. After amplification, the HBNF cDNA gene was cloned into pPIC9K, a shuttle expression vector for yeast system. The positive clone of expression vector bearing HBNF cDNA gene was obtained by screening. Verified recombinant vector was then used to transform Pichia strain GS115 by electroporation. His(+) transformants were selected on minimal dextrose medium (MD) plates which were histidine free. His(+) yeast recombinants with multi-copy inserts were screened in vivo by their resistance to G418. PCR analysis was used to confirm the integration of the HBNF cDNA gene into the Pichia genome. Secreted expression of hrHBNF protein in culture medium was obtained when the positive clone containing the HBNF cDNA gene was induced by methanol. The hrHBNF product purified by gel chromatography was added to cultured rat pheochromocytoma (PC12) cells to observe its ability to stimulate neurite outgrowth.</p><p><b>RESULTS</b>In the recombinant expression vector, the insert was sequenced to show exactly the sequence encoding human HBNF according to Genbank data. The HBNF cDNA gene was cloned downstream to the alpha-factor, and its open reading frame was in frame with the alpha-factor signal sequence in pPIC9K. SDS-PAGE showed that the molecular weight of the induced expression product was about 18 kDa, consistent with that of human HBNF reported in the literature. The protein product did promote neurite outgrowth in cultured rat pheochromocytoma (PC12) cells.</p><p><b>CONCLUSION</b>Recombinant human heparin-binding neurite-promoting factor can be expressed with a yeast system, and its product possesses the biological activity to promote neurite outgrowth.</p>
Subject(s)
Animals , Humans , Rats , Base Sequence , Carrier Proteins , Genetics , Pharmacology , Cytokines , Genetics , Pharmacology , DNA, Complementary , Chemistry , Genetic Vectors , Molecular Sequence Data , Neurites , Physiology , PC12 Cells , Pichia , Genetics , Recombinant Proteins , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Purpose To purify recombinant angiogenesis inhibitor r-K4K5 and investigate its inhibitoryeffects on bovine capillary endothelial (BCE) cell proliferation, chick embryo chorioallantoic membrane(CAM) angiogenesis and growth of experimental human non-small cell lung cancer (adeno). Methodsr-K4K5 was obtained by salting out and gel filtration with the purity of 95% determined by SDS-PAGE.BCE cells were cultured with DMEM media containing r-K4K5. The cells were counted in 24,48,72 hrespectively. r-K4K5 was injected daily into all 7-day chick embryo CAMs and CAM angiogenesis wasobserved at 72 h after incubation. The Balb/c (nu/nu) mice implanted with human SPC-Al tumor pieceswere grown for 10 days and then randomly divided into three groups. One group was treated with PBS, theother two groups were treated with local subcutaneous injection of purified r-K4K5 at 8 μg and 80 μg lpermouse every other day. They were daily observed and sacrificed in 14 days. Each tumor was weighed.Results The number of BCE cells, blood vessels diameter less than 50 μn of chick embryo CAM and theaverage weight of experimental tumor were decreased markedly in all the groups treated with r-K4K5.Conclusions r-K4K5 inhibits proliferation of BCE cells, angiogenesis of chick embryo CAMs and thegrowth of experimental human SPC-A1 non small lung cancer (adeno).
ABSTRACT
ObjectiveTo explore the inhibitory effects of r-k4k5 on retinal neovascularization.MethodsEighty-eight one-week-old C57BL/6J mice were put into the environment with 75% oxygen for 5 days to establish models of vascular proliferation retinopathy. One eye of each mouse received an intravitreal injection of 500 ng of r-k4k5 (large-dosage group) and of 250 ng of r-k4k5(small-dosage group), and the same volume of BSS was injected into the other eye of the mice both in these two groups as a control. The ADPase histochemical staining was used for retinal flatmount to observe changes of retinal vessels. The inhibitory effects of r-k4k5 on retinal neovascularization were evaluated by counting the endotheliocyte nuclei of new vessels extending from retina to vitreous in the tissue-slice.ResultsRegular distributions and reduced density of retinal blood vessels in eyes in the treatment group were found in retinal flatmount. The number of the endotheliocyte nuclei of new vessels extending from retina to vitreous was less in the eyes in the treatment group than which in control group (P