Optimization of transduction conditions of recombinant adeno-associated virus into NK92 cells / 中国药科大学学报

@#To improve the transduction efficiency of recombinant adeno-associated virus (rAAV) in NK92 cells, the number of cells, concentration of IL-2 in the medium, and serotype and dosage of rAAV were explored to optimize cell state and viral transfection conditions.Then, zinc chloride (ZnCl2), chloroquine, polyvinyl alcohol (PVA) and genistein with different concentration were added separately during transfection to further improve the viral transduction efficiency.The results showed that, at cell number of 5 × 105, the expression efficiency of enhanced green fluorescent protein (EGFP) was relatively high.When the IL-2 concentration was 1 000 IU/mL, NK92 cells were most suitable for virus transfection. The transduction efficiency of different serotypes of rAAV in NK92 cells was rAAV6, rAAV2 and rAAV9 in descending order.Pretreatment of NK92 cells with genistein could significantly increase the viral transduction efficiency, while the addition of other reagents had no significant effect.Through the optimization of the above conditions, the transduction efficiency of rAAV to NK92 cells could be significantly improved, which provided evidence for functional genetic modification of NK92 cells by rAAV.

Tropisms of rAAV2, 6, 8, 9 serotypes for intravitreal injection in the mouse retina / 中华实验眼科杂志

Objective:To compare the tropism of different adeno-associated virus (AAV) serotypes in retinal cells.Methods:The plasmids pFastBacDual-inCap and pFastBacDual-ITR-CMV-EGFP were constructed for AAV packaging with the baculovirus expression system.Recombinant adeno-associated virus type 2(rAAV2), 6, 8 and 9 serotypes were packaged, and the infectivity of rAAV was evaluated by infecting HEK293T cells at multiplicity of infection(MOI)2000.Twenty-five C57BL/6 mice were divided into five groups, with five mice per group.In the three experimental groups, both eyes of each mouse were injected 1 μl rAAV intravitreally, and 1 μl phosphate buffered saline (PBS) for the eyes of the control group.Two weeks after injection, the retinal tissues were collected for preparing flat mounts and cryosections.Enhanced green fluorescent protein (EGFP) gene expression was observed via fluorescence microscopy and laser scanning confocal microscopy.The study protocol was approved by the Ethics Committee of Suzhou Institute of Biomedical Engineering and Technology.Results:The infection efficiency of the recombinant virus to HEK293T cells was rAAV2>rAAV6>rAAV8>rAAV9, and the transduction efficiency was 39.5%, 18.4%, 8.7% and 4.6%, respectively.In mouse retinal transduction, rAAV2 and rAAV6 were highly expressed in the ganglion cells, and rAAV8 and rAAV9 were highly expressed in the retinal pigment epithelium (RPE) and photoreceptor cells.rAAV2-mediated EGFP expression in retinas was stable within three months after injection.Conclusions:Different rAAV serotypes have varying tropism and transduction efficiencies in retinal cells through intravitreal injection, rAAV2 has a high transduction efficiency and it can be stably expressed in retinas within three months after injection.

Ultra-sensitive quantification of the colorectal cancer-specific NDRG4 gene methylation levels in stool / 医学研究生学报

Objective The NDRG4 gene methylation in stool is a candidate biomarker for non?invasive diagnosis of colorectal cancer. However, the traditional methods for methylation detection could not be well applied to stool samples due to the low sensitivity and low specificity. The aim of this study was to develop a highly sensitive and specific method for quantifying the methylated NDRG4 gene in stools. Methods Forty one stool samples were collected from 12 colorectal cancer patients, 4 adenoma patients and 25 nor?mal persons. The invasive reaction was combined with real?time PCR and the relative quantification was performed by 2-ΔCT method to develop the highly sensitive and specific methylated DNA detection method, which was used for detecting NDRG4 methylation levels in 41 of stool samples. Results The sensitivity of the method was as low as 10 copies of methylated NDRG4 gene fragments. The specificity was high enough to distinguish 0.01% of methylated fragments from un?methylated fragments and 105 copies of unmethylated NDRG4 fragments gave noamplification signals. The detection results from 41 of stool samples showed that detection rate of the NDRG4 gene in stool from adenoma and colorectal cancer groups had a significant difference comparing to that from the normal group. Conclusion The 2-ΔCT method could accurately quantify the methylation levels of the NDRG4 gene in stool samples, and provide an efficient tool for non?invasive colorectal cancer detection.

Preparation of a novel AAV-ITR gene expression mini vector in Sf9 insect cells via baculovirus / 生物工程学报

AAV-ITR gene expression mini vector is a double-strand or single-strand DNA that only contains inverted terminal repeats of adeno-associated virus, cis-elements and gene of interest and does not contain any other foreign DNA sequences. We prepared Bac-ITR-EGFP and Bac-inrep. Spodoptera frugiperda cells were infected with Bac-ITR-EGFP (P3) and Bac-inrep (P3). Up to 100 μg of AAV-ITR-EGFP gene expression mini vectors were extracted from 2 x 10(7) cells of Sf9 72 h after infection. The gel electrophoresis analysis shows that most forms of AAV-ITR-EGFP gene expression mini vector were monomer and dimer. The mini vector expression efficacy was examined in vitro with HEK 293T cells. The EGFP expression was observed at 24 h after transfection, and the positive ratio reached 65% at 48 h after transfection.

Cloning, expressing of exendin-4 analogue and bioactivity analysis in vivo / 生物工程学报

To construct, express and purify Exendin-4 analogue and detect its biological activity in vivo. Insert gene sequence into fusion partner ofpED plasmid which is helped to purification, entitled the new recombinant plasmid 5 Exendin-4 analogue polypeptide gene and fusion partner gene was linked by acid hydrolysisgene, transformed to E. coli BL21 and the fusion protein was induced by lactose. After acid hydrolysis, the Exendin-4 analogue polypeptide separated from fusion chaperon. Anion charge chromatography were used to further purification. 6 to 8 week-old ICR mice were injected (s.c) with Exendin-4 analogue, blood glucose and plasma insulin level was detected in different period after oral glucose tolerance test. The results show that high expression of inclusion body was induced by lactose, which accounted for 40% of germ proteins, the Exendin-4 analogue was obtained with the purity of 91.8% after being purified by anion charge chromatography. Bioactivity assay showed that the level of blood glucose of mouse which treated with exendin-4 analogue was obviously decreased to normal (P < 0.01), and the level of plasma insulin was increased obviously (P < 0.01).

Improved efficacy of P277 fused to heat shock protein 65 of Mycobacterium tuberculosis against diabetes in nonobese diabetic mice / 生物工程学报

To improve the efficacy of peptide P277 in preventing autoimmune diabetes, heat shock protein 65 kD (HSP65) of Mycobacterium tuberculosis var. bovis was fused with linear polypeptide epitope of P277 and expressed as soluble protein in Escherichia coli. The fusion protein HSP65-P277 was purified by anion exchange column chromatography and then used to immunize prediabetic NOD mice with three ip inoculations in absence of adjuvants. Serum samples from the immunized mice were collected monthly and the concentration of blood glucose was measured. The study showed that administration of HSP65-P277 to NOD mice could prevent the development of diabetes more efficiently than the peptide P277 itself or HSP65. Fused to heat shock protein 65 of Mycobacterium tuberculosis could improve the efficacy of diabetes prevention of P277 in nonobese diabetic mice. The results suggest the fusion protein of HSP65-P277 would be useful for treating insulin-dependent diabetes mellitus.

Effect of Displaying P277 Peptide on Surface of L-asparaginase on Its Antigenicity and Efficacy of Autoimmune Diabetes Prevention in NOD Mice / 中国生物化学与分子生物学报

The recombinant chimeric enzyme of AnsB-TTP-P277 comprising L-asparaginase, a tetanus toxin peptide spacer and P277 was expressed as a soluble protein in Escherichia coli. The purified chimeric enzyme exhibited primary activity of the native asparaginase. Prediabetic NOD mice immunized with the chimeric enzyme could induce specific antibodies against P277 and the specificity of anti-P277 antibodies was verified by Western blot assay. The study showed that displaying the P277 epitope on the surface of asparaginase could effectively overcome the weak antigenicity of the P277 epitope and evoke a strong P277-specific immune response in mice. Moreover, the concentration of blood glucose was measured by an automated analyzer.Histochemical analysis of mice pancreas tissues showed that the administration of the chimeric enzyme to NOD mice could prevent the development of diabetes more efficiently than the peptide P277 itself.