[Construction of camelidae natural nanobody phage dispaly library and screening, production and identification of anti-CD19 nanobody].

Objective To construct and verify camelidae natural nanobody phage display library for selection of nanobodies against various antigens, and to obtain and identify the nanobody targeting CD19. Methods The total RNA of spleen of Bactrian camel was reverse transcribed and the variable region gene fragment of its heavy chain was obtained by nested PCR. It was constructed into the pCANTAB5e phagemid vector and electrotransformed into TG1 E. coli to develop the natural nanobody phage display library. After rescued by the KM13 helper phage, its capacity and diversity were analyzed and identified. Nanobody against CD19 was screened using biotinylated antigen combined with streptavidin magnetic beads, followed by ELISA, sequencing, exogenous expression and verification. Results The constructed natural phage nanobody display library had great diversity, and its fragment insertion rate was about 100%. The amino acid homology of 20 randomly selected clones was 65.85%, and the titer of the display library rescued by the helper phage was 9.0×1013 CFU/mL. After panning with CD19 as the antigen, the positive clones were sequenced and analyzed, and finally anti-CD19 nanobody sequences were obtained. The exogenously expressed anti-CD19 nanobody based on the sequences was verified having the ability to bind to CD19. Conclusion A camelidae natural nanobody phage display library with high titer and great diversity has been successfully constructed. Three anti-CD19 nanobody sequences have been obtained by panning with CD19. In addition, this study provides technical support for researching and developing diagnostic kits and antibody drugs targeting CD19, and it is a novel direction to improve CAR-T cells targeting CD19.

[Optimization of CAR-T cell culture system and lentivirus transduction conditions].

Objective To optimize the culture system of chimeric antigen receptor T (CAR-T) cells in vitro and lentivirus infection conditions. Methods Peripheral blood mononuclear cells (PBMCs) of healthy people and umbilical cord blood mononuclear cells (UCBMCs) of healthy pregnant women were isolated and purified by CD3 magnetic beads, and then they were cultured in different cell culture systems. There were eight cell culture systems containg different combinations of the following components: recombinant human interleukin 2 (rhIL-2), rhIL-12, rhIL-18, rhIL-7, rhIL-21, TWS119. Cell proliferation was detected by counting the cells at 0, 3, 5, 7, 10, 18 days after the cells were seeded into cell plates. Flow cytometry was used to detect the expression of programmed death 1 (PD-1), and ELISA was used to detect the expression of interferon-γ (IFN-γ). Cell culturing plates were coated with serial concentrations of recombinant human fibronectin fragment (RetroNectinr) (0, 20, 50 µg/mL), and antibodies against human CD3/CD28 (250, 500, 1 000 ng/mL). Then T cells cultured in the above plates were infected with negative control lentivirus at different multiplicity of infection (MOI=3, 5); 72 hours later, expression of green fluorescent protein (GFP) was observed under a fluorescence microscope to preliminarily determine virus infection efficiency. Flow cytometry was used to detect CD3/GFP positive rate to obtain lentivirus infection conditions. CD19 CAR lentivirus was packaged. Real-time quantitative PCR and Western blotting were performed to detect whether the CD19 CAR vector was successfully constructed. Finally, T cells were cultured in 1 µg/mL anti-human CD3/CD28 and 20 µg/mL RetroNectinr-coated culture plates, and rhIL-2, rhIL-12, rhIL-18 were added in the culture medium, then the cells were infected with CD19 CAR lentivirus at the optimized virus infection conditions. Results The cell culture system with the best proliferation ability was rhIL-2 combining with rhIL-18; the cell culture system with the strongest release of IFN-γ was rhIL-2 and rhIL-12 combined with rhIL-18. When the dose of antibodies against CD3/CD28 was 1 µg/mL, RetroNectinr was 20 µg/mL, and MOI was 3, the virus infection efficiency was optimal. The positive rate of CAR-T cells was 34% under the optimal condition. Conclusion The study achieved the optimal cell culture system of CD19 CAR-T cells in vitro and the conditions of lentivirus infection on primary T cells.

RNA-sequencing analysis reveals betalains metabolism in the leaf of Amaranthus tricolor L.

Amaranth plants contain large amounts of betalains, including betaxanthins and betacyanins. Amaranthin is a betacyanin, and its molecular structure and associated metabolic pathway differ from those of betanin in beet plants. The chlorophyll, carotenoid, betalain, and flavonoid contents in amaranth leaves were analyzed. The abundance of betalain, betacyanin, and betaxanthin was 2-5-fold higher in the red leaf sectors than in the green leaf sectors. Moreover, a transcriptome database was constructed for the red and green sectors of amaranth leaves harvested from 30-day-old seedlings. 22 unigenes were selected to analyze the expression profiles in the two leaf sectors. The RNA-sequencing data indicated that many unigenes are involved in betalain metabolic pathways. The potential relationships between diverse metabolic pathways and betalain metabolism were analyzed. The validation of the expression of 22 selected unigenes in a qRT-PCR assay revealed the genes that were differentially expressed in the two leaf sectors. Betalains were biosynthesized in specific tissues of the red sectors of amaranth leaves. Almost all of the genes related to betalain metabolism were identified in the transcriptome database, and the expression profiles were different between the red sectors and green sectors in the leaf. Amaranth plants consist of diverse metabolic pathways, and the betalain metabolic pathway is linked to a group of other metabolic pathways.