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Objective To explore the method to develop a gene vaccine of human cytomegalovirus (HCMV) phosphoprotein 65 (pp65) against its infection. Methods HCMV strain AD169 was propagated in WI-38 cell and viral DNA was extracted as a template for polymerase chain reaction (PCR) amplification of UL83 (pp65), the resulting of PCR was subcloned into pUC118HincII/BAP plasmid and DNA sequence analysis conformed the fidelity of the PCR. The vector pcDNA 5.0 was designed to correctly place CMV promoter sequence, pp65 sequence and secret signal sequence (mouse immunoglobulin kappachain for efficient secretion of recombination protein) into its genomic DNA. Exchanged primers of pp65 sequence, CMV promoter sequence and secret signal sequence to confirm the result by PCR screening. The vector pcDNA 5.0 was transfected into CHO cell, supernatants of transfected cells were extracted and purified. Recombination protein from supernatants was detected by gel electrophoresis and dot blot hybridization of Western- ECL system. Results Compared the sequence of pp65 gene with the standard sequence of pp65 from Medline,it was found that the concordant rate between them was 99.99%,only a nonsense mutation occurrences at 1 455 base.A pcDNA 5.0 Eukaryotic expression vector was established, which including CMV promoter sequence,secretion signal sequence and pp65 sequence. PCR screening and the pp65 protein expressed in CHO cell confirmed it. Extraction from supernatants in transfected CHO cells was recombination protein of pp65, which was detected by gel electrophoresis and dot blot hybridization of Western- ECL system and western blotting.Conclusion Subunit vaccine of HCMV is gained,which is a transfer eukaryotic expression vector pcDNA 5.0 constructed by CMV promoter sequence,secretion signal sequence and pp65 gene sequence.

9.Migration and Neuron Differentiation of Neural Stem Cells When Transplanted to the Lateral Ventricle of Neonatal Rat

xiao-song, BU ; zuo, LUAN ; guo-cai, YIN ; jiu-lai, TANG

Journal of Applied Clinical Pediatrics 2006;0(24):-

Objective To explore the migration and differentiation of the human neural stem cells (hNSC) after being transplanted to the neonatal rat lateral ventricle,to provide some data on therapy for neonatal cerebropathy by using of neural stem cells.Methods N2 medium containing EGF+FGF2+LIF was used to culture the NSC spheres from the forebrain tissues of aborted human fetus.The hNSC was identified by detecting the NSC marker nestin antigen and showing the potency to differentiate into neural cells( including astrocytes,oligodendrocytes and neurons)by using indirect immuno-fluorescence assay(IFA).The part of the hNSC in-vitro cultured for 14 d was digested to suspensions of cell.Cultured for 14 d, the hNSC in-vitro and the suspension were transplanted into the lateral ventricles of the neonatal rat brains.The rats were respectively killed at 24,48 and 72 h respectively post-transplant,the whole brain was sectioned,and the special immuno-response detection was performed by using anti-human nuclei(anti-hNuc)and anti-human neurofilament(anti-hNF).Results In vitro culture,the typical NSC spheres were obtained from the forebrain of the human fetus.The suspensions of cells were obtained from the neurosphere.In neurosphere group, the results of anti-hNuc detecting tracing at 72 h post-injection showed that the grafts had migrated into the cortex grand layers of olfactory bulbus,medial precentral area of lobus frontalis,hippocampal,and lobus occipitalis.The label-positive cells lined along the Cerebellar Purkinje cell layers and appeared in most parts of mesencephalons.The immuno-respons results of anti-hNF showed that the positive cells scattered in the grand layer of cortex,the connection among positive cells was watched.In suspensions group,the results of anti-hNuc detecting tracing at 24 h post-injection show a great quantity of positive cells in the ventricles and injection track.At 72 h, a small quantity of positive cells remained in the ventricle and nearby brain tissue.Conclusions Whole neurospheres migrated intensely and differentiated into neurons and gliocytes.At the same time,transplants of cells from suspension transplants showed limited or no migration because of internal environment of the brain and construction of neurospheres.