人载脂蛋白AⅠ基因的克隆表达及功能

To identify, clone ,sequence and highly express the mature peptide gene of ApoA Ⅰ, total RNA was prepared from human fetal liver tissue. cDNA fragment encoding human ApoA Ⅰ was amplified by RT-PCR using specific primers, and then was inserted in pGEM-T vector. DNA sequencing indicates that the fragment is 729 base pairs in length and has 100% nucleotide homology with that of reported ApoA Ⅰ cDNA gene previously. The ApoA Ⅰ gene was cloned into pGEX 5X-1.The recombinant protein was expressed in E.coli DH5α, purified by glutathione-Sepharose 4B affinity chromatography and confirmed by SDS-PAGE. It was shown that the recombinant ApoA Ⅰ was expressed in E.coli, and the target protein amounted to 36% of total bacteria proteins. Cholesteryl ester transfer experiment showed that the recombinant ApoA Ⅰ was capable of promoting transfer of CE from HDL to LDL. Western blotting showed that the protein could react specifically with anti-ApoA Ⅰ antibodies.     

表达鸡传染性支气管炎病毒JS/95/03株S1蛋白的重组杆状病毒构建

The recombinant baculovirus expressing S1 glycoprotein of nephropathogenic strain JS/95/03 of infectious bronchitis virus was generated by using the Bac-to-Bac baculovirus expression system.The BamH I-Sal Ⅰ fragment containing S1 gene from the recombinant plasmid pMDJS9503S1 was purified and cloned in frame into the baculovirus transposing vector pFASTBAC HTa under the polyhedrin gene promoter.The recombinant transposing plasmid pFASTJS9503S1 was screened and then transformed into Escherichia coli DH10BAC.The resulting recombinant bacmid rBacmidJS9503S1 was transfected into cells of the insect Spodoptera frugiperda(Sf9)and the recombinant baculoviruse rAcJS9503S1 was obtained.The lysates of cells infected with rAcJS9503S1 were analyzed by SDS-PAGE and the expressed product of S1 gene was detected by Western bloting and immunofluorescence assay(IFA).The results showed the recombinant baculovirus was fully capable of expressing S1 glycoprotein of JS/95/03.Maybe owing to the incomplete glycosylation in insect cells,the S1 gene product had a Mr of only 61000.In immunofluorescence test and Western blotting,the expressed product could react with polycolonal antibody against IBV M41 strain,indicating it possessed the antigenic properties specific for native S1 glycoprotein.     

High Level Expression of Grass Carp Reovirus VP7 Protein in Prokaryotic Cells

Sequences analysis revealed Grass carp reovirus （GCRV） s10 was 909 nucleotides coding a 34 kDa protein denoted as VP7, which was determined to be a viral outer capsid protein （OCP）. To obtain expressed OCP in vitro, a full length VP7 gene was produced by RT-PCR amplification, and the amplified fragment was cloned into T7 promoted prokaryotic expression vector pRSET. The recombinant plasmid, which was named as pR/GCRV-VP7, was then transformed into E.coli BL21 host cells. The data indicated that the expressed recombinant was in frame with the N-terminal fusion peptide. The over-expressed fusion protein was produced by inducing with IPTG, and its molecular weight was about 37kDa, which was consistent with its predicted size. In addition, the fusion protein was produced in the form of the inclusion body with their yield remaining steady at more than 60% of total bacterial protein. Moreover, the expressed protein was able to bind immunologically to anti-his-tag monoclonal antibody （mouse） and anti-GCRV serum （rabbit）. This work provides a research basis for further structure and function studies of GCRV during entry into cells     

Cloning of M and NP Gene of H5N1 Avian Influenza Virus and Immune Efficacy of their DNA Vaccines

The M and NP genes of H5N1 avian influenza virus (A/chicken/Hubei/489/2004) were amplified by RT-PCR from viral RNA, and cloned into pMD18-T vector respectively. The expression plasmid containing the M gene (pHM6-m) or the NP gene (pHM6-np) was then constructed by inserting the M or NP gene into the pHM6 eukaryote expression vector; the constructed plasmid was then sequenced. 32 BALB/c mice (6-week-old) were divided into four groups at random. Three groups of BALB/c mice were inoculated one time the intramuscular route with either 30 ug of plasmid pHM6-m, 30μg of plasmid p HM6-np or the mixture of plasmid pHM6-m (15μg) and pHM6-np( 15μg) respectively. A additional group of mice were injected with 100μl PBS as controls. Two weeks later, all mice were challenged with homologous H5N1 avian influenza virus, and observed in the following 12 days. The survival rates of mice in the pHM6-m group, the pHM6-np group and mixed plasmids group were 62.5%, 25.0% and 50.0%, respectively. Results showed that effective protection could be provided by either pHM6-m or pHM6-np, but pHM6-m provided a better protective effect than pHM6-np.     

马铃薯Y病毒N株HC-pro基因的序列分析与表达

The HC-pro gene was amplified by RT-PCR from total RNA of tobacco leaves infected with a N strain of Potato virus Y in Shaanxi, and cloned into the PMD 18-T vector. This HC-pro gene is consisted of 1371 nucleotides, encoding 457 amino acids. It shared the sequence homologyof 82.5%-96.4% nucleotide acid and 92.5%-98.0% in amino acids compared to 9 species of PVY N HC-pro abroad. The HC-pro gene was inserted into prokaryotic expressing vector pBV221, to obtain pBVHC recombinant plasmid in E. coli BL21. SDS-PAGE indicated that HC-pro proteins are successfully expressed in E. coli, Western blotting analysis demonstrated that the antibody against the expressed HC-pro can be used to identify the infected plants .     

Subcellular Localization Analysis of Bovine Foamy Virus Borfl Protein

The Borfl protein is encoded by an immediate-early gene of the bovine foamy virus （BFV） and plays a key role in the viral life cycle. Borfl is a DNA binding protein which can transactivate both the long terminal repeat （LTR） and the internal promoter （IP） of BFV by specifically binding to the transactivation responsive element （TRE）. To analyze the subcellular localization of Borfl during the BFV life cycle, this gene was cloned into a prokaryotic expression vector and expressed in a soluble form. After the purification and immunization, we raised the mouse anti-Borfl serum with a high titer based on ELISA results. Western blot analysis showed that the antiserum could specifically recognize the Borfl protein that was expressed in 293T cells. With this specific serum, we revealed the nuclear and cytoplasmic localization of Borfl in HeLa cells that was transfected with Borfl. Moreover, the immuno-fluorescence assay also showed that the localization of Borfl during the infection and transfection of BFV was identical.     

Characteristics of the membranes of the pellicle of the ciliatePseudomicrothorax dubius

Summary The membranes of the pellicle of the ciliatePseudomicrothorax dubius are investigated using thin section electron microscopy and freeze-fracture replicas. The plasma membrane is covered by a surface coat and is connected to the outer alveolar membrane by short, sometimes branched, bridges. The inner alveolar membrane is coated on both sides. The epiplasm lies in intimate contact with the cytoplasmic surface of this membrane, and there is a corresponding deposit on the other surface. This deposit is regularly striated.The epiplasmic layer and the alveoli are interrupted at sites of cytotic activity,e.g., the attachment sites of trichocysts, the cytoproct, and the parasomal sacs. The striated deposit ends where the epiplasm ends, indicating a direct relationship between these two epimembranous layers.There is a deposit along the sides of the first part of the tip of the trichocysts, and in this region the trichocyst membrane is free of intramembranous particles.The membrane of the parasomal sacs has a coat on both surfaces. That on the extraplasmic surface is similar to the surface coat of the plasma membrane. The origin of the cytoplasmic coat is unknown. The cytotic activity of these sacs is indicated by their highly irregular profiles.     

The early development of the tail and the transformation of the shape of the nucleus of the spermatid of the domestic fowl,Gallus gallus

Summary The differentiation of the spermatid, especially in reference to the formation of the flagellum, and transformation of the shape of the nucleus was investigated in the domestic fowl.In the early stage of the spermatid, a prominent Golgi apparatus appears around the centrioles. The Golgi vesicles then surround the axial-filament complex which develops from the distal centriole. These vesicles fuse to form continuous membrane at the earliest stage of flagellar formation, and in the succeeding stage Golgi lamellae are attached to the plasma membrane of the developing flagellum. From these observations, it is assumed that Golgi apparatus may be a source of the membrane system of the flagellum.The microtubules distributed around the nucleus form the circular manchette. The anterior region of the nucleus with the manchette is cylindrical in shape and the posterior region without it remains irregular in shape. When the circular manchette has been completed, the whole nucleus acquires a slender cylindrical shape. The circular manchette then changes into the longitudinal manchette. The nuclei of spermatids without a longitudinal manchette are abnormal in shape. In view of these observations it is assumed that the nuclear shaping of the spermatid may be accomplished by circular manchette and the maintenance of shape of the elongated nucleus by longitudinal manchette.The authors wish to thank Mr. Takayuki Mori for his helpful suggestions and technical advices     

Characterization of the composition of the aqueous humor and the vitreous body of the eye of the frog Rana temporaria L

This study aimed to analyze the aqueous humor (AH) and the vitreous body (VB) of the eye of the adult frog Rana temporaria L. as a representative species of amphibians, which lead a semi-terrestrial life. The presence of collagen, albumin, uric acid and electron donors was shown in both media; however, there are slight differences in their concentrations. To determine collagen, a spectral-fluorescent probe, cyanine dye, was used. The presence of collagen in AH of the frog was found at the first time. The total content of electron donors (ascorbic and uric acids, tryptophan, and tyrosine) in VB and HA was roughly estimated at ~ 1.5 × 10^− 4 mol/L. Both VB and AH absorb light in similar UV regions. The total protein and albumin contents in AH were found to be somewhat higher than those in VB. The uric acid content was at an equally low level in both intraocular media. It is supposed that the similarity of VB and AH compositions shown in this work is due to some exchange between VB and AH contents in the course of accommodation. The role of intraocular fluids in physiological functions of the eye and in protecting the retina against UV light is discussed.     

Observations on the permeability of the choriocapillaris of the eye

Summary The choriocapillaris is a fenestrated capillary bed located posterior to the retinal pigment epithelium. It serves as the main source of supply to the photoreceptors, retinal pigment epithelium, and other cells of the outer retina. The permeability of these capillaries to intravenously injected ferritin (MW — approx. 480,000; mol. diam. 11 nm) was examined in the mouse, rabbit, and guinea pig, each of which is characterized by a different type of retinal vascularization. In all three species, the bulk of the ferritin remained in the capillary lumina, where it appeared to be blocked at the level of the diaphragmed fenestrae. Some ferritin was present in endothelial cell vacuoles. The results confirm previous work on the rat choriocapillaris and indicate that the barrier function of the choriocapillary endothelium is present even among species in which the retinal circulation differs significantly.Supported by NIH grant EY03418