Rescue of an Enterotropic Newcastle Disease Virus Strain ZM10 From Cloned cDNA and Stable Expressing an Inserted Foreign Gene

Background: Newcastle disease virus (NDV) strain ZM10, a typical enterotropic avirulent vaccine strain, has been widely used in in China for chickens against Newcastle disease. To elucidate its enterotropic mechanism and develop recombiant multivalent vaccines based on it, the reverse genetics system for NDV ZM10 is an indispensable platform.Results: A full-length cDNA clone of NDV ZM10 and three supporting plasmids were constructed using the ligation-independent cloning (LIC) method. Recombinant NDV rZM10 was successfully rescued after these plasmids were co-transfected into BHK-21 cells. Besides, the recombinant virus rZM10-RFP encoding the red fluorescent protein was generated by inserting the RFP gene into the full-length clone of NDV between the P and M genes. These rescued viruses were genetically and biologically identical to the parental strain and showed similar growth kinetics. Conclusion: The recovery system of NDV ZM10 strain was established, and can be used as a foundation for research on the enterotropic mechanism and development of multivalent vaccines against viral diseases of livestock and poultry.

ISOLATION AND STRUCTURE ANALYSIS OF THE MUTANT FORM OF N-TERMINAL CATALYTIC MODULE OF BOS TAURUS TYROSYL-tRNA SYNTHETASE WITH REPLACEMENT OF Trp 40 AND Trp 87 BY ALANINE

Aim. Isolation and analysis of the structure of the mutant monotryptophan protein mini BtTyrRS for study of conformational changes of the enzyme at the stage of interaction with tRNA using fluorescence spectroscopy and determination of the effect of tryptophan residues in position 40 and 87 in its structure on the functional properties of the enzyme. Methods. Electrophoresis, metal-chelating affinity chromatography, fluorescence spectroscopy, spatial structure modeling. Results. It was found that the replacement of two codons of Trp by codons of Ala in the cloned cDNA mini BtTyrRS does not affect the synthesis of the mutant form of the enzyme in E. coli strain BL21 (DE3) pLysE. The yield of affinity purified protein on Ni-NTA agarose is on average 3.5 mg per 100 ml of culture medium. Computer modeling of the structure and fluorescence spectroscopy of the monotryptophan form of mini BtTyrRS indicates a compact structure of the mutant enzyme, in which Trp 283 is in an immobilized microenvironment. Conclusions. Affinity purified on Ni-NTA agarose mutant monotryptophan protein mini TyrRS have been obtained which is suitable for fluorescent studies of structural-dynamic and functional properties of the enzyme.

Dual Promoters Improve the Rescue of Recombinant Measles Virus in Human Cells

Reverse genetics is a technology that allows the production of a virus from its complementary DNA (cDNA). It is a powerful tool for analyzing viral genes, the development of novel vaccines, and gene delivery vectors. The standard reverse genetics protocols are laborious, time-consuming, and inefficient for negative-strand RNA viruses. A new reverse genetics platform was established, which increases the recovery efficiency of the measles virus (MV) in human 293-3-46 cells. The novel features compared with the standard system involving 293-3-46 cells comprise (a) dual promoters containing the RNA polymerase II promoter (CMV) and the bacteriophage T7 promoter placed in uni-direction on the same plasmid to enhance RNA transcription; (b) three G nucleotides added just after the T7 promoter to increase the T7 RNA polymerase activity; and (c) two ribozymes, the hairpin hammerhead ribozyme (HHRz), and the hepatitis delta virus ribozyme (HDVrz), were used to cleavage the exact termini of the antigenome RNA. Full-length antigenome cDNA of MV of the wild type IC323 strain or the vaccine AIK-C strain was inserted into the plasmid backbone. Both virus strains were easily rescued from their respective cloned cDNA. The rescue efficiency increased up to 80% compared with the use of the standard T7 rescue system. We assume that this system might be helpful in the rescue of other human mononegavirales.

An Experimental Approach to Rigorously Assess Paneth Cell α-Defensin (Defa) mRNA Expression in C57BL/6 Mice

Abundant evidence from many laboratories supports the premise that α-defensin peptides secreted from Paneth cells are key mediators of host-microbe interactions in the small intestine that contribute to host defense and homeostasis. α-defensins are among the most highly expressed antimicrobial peptides at this mucosal surface in many mammals, including humans and mice; however, there is striking variation among species in the number and primary structure of α-defensin paralogs. Studies of these biomolecules in vivo are further complicated by striking variations between laboratory mouse strains. Herein, we report an experimental approach to determine with precision and specificity expression levels of α-defensin (Defa) mRNA in the small intestine of C57BL/6 mice through an optimized set of oligonucleotide primers for qRT-PCR assays and cloned cDNA plasmids corresponding to the Defa paralogs. This approach demonstrated marked differences in α-defensin expression in C57BL/6 mice with respect to proximal/distal anatomical location and developmental stage, which have not been described previously. These data underscore the importance of careful attention to method (primer choice, proximal vs. distal location, and developmental stage) in analysis of antimicrobial peptide expression and their impact.

Structure, Enzymatic Mechanism of Action, Applications, Advantages, Disadvantages and Modifications of Luciferase Enzyme

North American Firefly, Photinu spyralis, entertainment countless observers, probably because of human arrival mainland. Between the period of 1950s and 1980s, many young biologists spent their money as a firefly collector, first hired by a Professor William D McElroy at Johns Hopkins University, later became a member of the Sigma Firefly Club. In 1985, when Marlene DeLuca and her colleagues cloned cDNA encoding luciferase, a replacement source of the enzyme becomes available and later in labs around the world, many other organisms began to emit unique yellow-green glow as a result of firefly expression Luciferase is in their cells. Now, luciferase and its genes have become very useful for research purposes and also for a variety of commercial purposes. The structure of firefly and bacterial luciferase will be of great value for the development of applications in many processes. There are many firefly luciferase homologues that can catalyze similar reactions with similar amino acid sequences. Several applications are described in different publications on bioluminescence and chemiluminescence. Firefly flicker is always very interesting to observe it. Firefly luciferase emits light but does not generate heat, which also causes curiosity because it solves the enzyme-catalyzed reaction. Here we mainly review the structure, mechanism, application, advantages and disadvantages of luciferase.

Molecular cloning of an S-adenosylmethionine synthase gene from pigeon pea (Cajanus cajan) and its expression analysis under arbuscularmycorrhizae fungi colonization and drought stress

An S-adenosylmethionine synthase (SAMS) gene associated with the drought responsiveness was isolated and characterized from pigeon pea. It was designated CcSAMS and contained an open reading frame of 1,182 bp, which encoded 394 amino acid residues. Sequence analysis of the cloned cDNA showed 94% identity with SAMS from other plant species, suggesting that this gene was considerably conserved in plants. Gene expression analysis demonstrated that CcSAMS was highly expressed in the leaves of AM-colonized plants, irrespective of exposure to either drought or drought-free. Rather, the expression levels of AM plants were significantly higher than that of NAM plants as subjected to drought stress. Therefore, AM symbiosis might enhance the expression of CcSAMS, and the elevated tolerance of AM- colonized pigeon pea to drought-stress was at least partially ascribed to the overexpression of SAMS gene.