The Characteristics of RNA Vaccine; its Strengths and Weaknesses

Since 1990 when Wolff and co-authors proved that both RNA and DNA expression vectors containing interest gene were directly injected into mouse muscle and expressed the protein in vivo, the concept of gene vaccine has been broadly tested in the vaccine field. However, due to the limitations of technology and the misconception about RNA, most previous studies have focused on the DNA vaccine. Recently, the RNA vaccine has emerged as a new game-changing disruptive innovation technology in the vaccine field. This review has covered the characteristics of the RNA vaccine, including its strengths and weaknesses. Finally, we have suggested future directions for the RNA vaccine.

Establishment of a rapid method for synthesis and detection of gRNA / 中国实验动物学报

Objective The purpose of this study was to establish a rapid method for synthesis and detection of guild RNA (gRNA) which is an essential component in CRISPR/Cas9 knockout technology .Methods First, the Nkp46 gRNA core fragment was synthesized as amplification template .Second , the forward and reversed primers of the matched gRNA were designed using the Nkp46 gene as reference sequence .Third, the DNA fragment of Nkp46 gRNA was amplified by PCR technology using the synthesized gRNA core fragment as template .The gRNA was reversely transcribed in vitro u-sing amplified DNA fragment as template .The efficiency and specificity of gRNA and its interaction with Cas 9 were detec-ted in vitro.Results The specificity and activity of Nkp46 gRNA were high .The obtained gRNA interacted with Cas 9 en-zyme and successfully cut the target double-stranded DNA at the designed site .Conclusions The method for synthesis and detection of gRNA established in this study is faster than the original method , and the created gRNA is fully functional .

Preparation and purification of siRNA targeting a proliferation-inducing ligand of pancreatic cancer cell line / 第二军医大学学报

Objective: To prepare and purify siRNA targeting a proliferation-inducing ligand targeted (APRIL-siRNA), so as to provide a basis for studying the role of APRIL in human pancreatic cancer. Methods: pET-22b-APRIL was constructed to express APRIL dsRNA of human pancreatic cancer cell line CFPAC-1 in E. coli and the product was purified by chromatography using CF-11 column. APRIL dsRNA was digested by RNase III to prepare APRIL siRNA, then the reaction mixture was loaded onto a DEAE ion exchange chromatography to remove RNase III from oligonucleotides, and size exclusion chromatography was used to purify 21 bp siRNA. The purified APRIL siRNA was used to transfect Chinese hamster ovary (CHO) cells and the expression of APRIL in CHO cells was observed under fluorescence microscope. Results: APRIL dsRNA was successfully expressed in E. coli after IPTG induction and was purified by CF-11 column. dsRNA was hydrolyzed with RNase III and was purified by DEAE ion exchange chromatography and size exclusion chromatography. 15% nondenaturing PAGE and 12% SDS-PAGE confirmed that RNase III was removed from oligonucleotides and 21 bp siRNA was purified with size exclusion chromatography. It was also found that APRIL siRNA obviously depressed APRIL expression in CHO cells. Conclusion: We have successfully constructed APRIL siRNA targeting APRIL gene of CFPAC-1 cells with in vitro transcription, which provides a basis for knock-down of APRIL gene in CFPAC-1 cells.

Observation of chimeric hepatitis C virus In infected Huh7.5 cell through transmission electron microscopy / 中华微生物学和免疫学杂志

Objective To observe the morphological characteristics of HCV particles and intracel-lular ultrastructure changes in Huh7. 5 cells which was infected with chimeric HCV via transmission electron microscopy. Methods Plasmid J6/JFH encoding the full length HCV chimeric genome was transcribed to HCV RNA in vitro and the RNA was transfected into Huh7.5 cells by electroporation. Quantitative real-time PCR(qRT-PCR) was used to assay HCV copies of the supernatant of transfected cells. Indirect immunofluo-rescence was used to detect the expression of HCV proteins. The cell culture superoatant were used to infect narve Huh7.5 cells, transmission electron microscopy was used to observe morphological characteristics of vi-rus particles and intracellular ultrastructure changes in infected Huh7. 5 cells. Results qRT-PCR showed high level virus copies in supernatant of transfected cells collected in different times, indirect immuno-fluo-rescencc proved high expression of HCV NS5A proteins in the transfected cells. Large numbers of enveloped or unenveloped virus-like particles (VLPs) were observed in infected Huh7. 5 cells via transmission electronmicroscopy. We also found hyperplasia of some membrane-enclosed organelles in the cytoplasm. Several fea-tures characterizing flavivirus infected cells and a cytoplasmic inclusion of unknown origin were observed. Conclusion The chimeric HCV from in vitro cell culture system is proved to be intact virus particles which can efficiently infect Huh7.5 cells.

Preparation and purification of siRNA targeting a proliferation-inducing ligand of pancreatic cancer cell line / 第二军医大学学报

Objective:To prepare and purify siRNA targeting a proliferation-inducing ligand targeted(APRIL-siRNA),so as to provxde a basis for studying the role of APRIL in human pancreatic cancer.Methods:pET-22b-APRIL was constructed to express APRIL dsRNA of human pancreatic cancer cell line CFPAC-1 in E.coli and the product was purified by chromatography using CF-11 column.APRIL dsRNA was digested by RNaseⅢto prepare APRIL siRNA,then the reaction mixture was loaded onto a DEAE ion exchange chromatography to remove RNaseⅢfrom oligonucleotides,and size exclusion chromatography was used to purify 21 bp siRNA.The purified APRIL siRNA was used to transfect Chinese hamster ovary(CHO)cells and the expression of APRIL in CHO cells was observed under fluorescence microscope Results:APRIL dsRNA was successfully expressed in E.coli after IPTG induction and was purified by CF-11 column.dsRNA was hydrolyzed with RNaseⅢand was purified by DEAE ion exchange chromatography and size exclusion chromatography.15% nondenaturing PAGE and 12% SDS- PAGE confirmed that RNaseⅢwas removed from oligonucleotides and 21 bp siRNA was purified with size exclusion chromatography.It was also found that APRIL siRNA obviously depressed APRIL expression in CHO cells.Conclusion:We have successfully constructed APRIL siRNA targeting APRIL gene of CFPAC-1 cells with in vitro transcription,which provides a basis for knock-down of APRIL gene in CFPAC-1 cells.

Expression of human IL-2 receptor chain cDNA in vitro transcripts / 中国免疫学杂志

Recombinat plasmid pJSB13 has an insert of 121-937 bp of human IL-2R_? cDNA in multiple cloning site of pGEM3 vector.This plasmid was digested with EcoR I to linearize it and then in vitro transcripted with SP6 RNA polymerase and capped with cap structure analog 7mG (5') G.The capped transcripts were translated in rabbit reticutocyte lysate or Xenopus Iaevis oocytes respectively. The results demonstrated that they can be efficiently translated as functional mRNA in both systems. Moreover, the translation product, IL-2R_?,can be secreted into oocvte culture medium.The program we had used enables one to produce and identify mRNA and conse quently protein from any cDNA clone. Furthermore this research can be of much helpful to the researches on the relationship between structure, function and expression of IL-2R_?.