Construction and Immunogenicity of a Recombinant Pseudorabies Virus Expressing SARS-CoV-2-S and SARS-CoV-2-N.

Coronavirus (CoV) is an important pathogen of humans and animals, which can infect humans or animals through the respiratory mucosal route. Syndrome coronavirus 2 (SARS-CoV-2) is quite similar to syndrome coronavirus (SARS-CoV) with the same receptor, angiotensin-converting enzyme 2 (ACE2). The S and N proteins are the most important protective antigens of the SARS-CoV-2. The S protein on the viral membrane mediates the virus attachment with the host cells, and the N protein is the most abundant expression during infection. In this study, the recombinant viruses expressing the S and N proteins of SARS-CoV-2 were successfully constructed by Red/ET recombinant technology using Pseudorabies virus (PRV) strain Bartha-K61 as a vector. Genetic stability and growth kinetics analysis showed that the recombinant viruses rPRV-SARS-CoV-2-S and rPRV-SARS-CoV-2-N had similar genetic stability and proliferation characteristics to the parental PRV. The immunoassay results showed that mice immunized with recombinant viruses could produce total IgG antibodies. Therefore, PRV is feasible and promising as a viral vector to express SARS-CoV-2-S and SARS-CoV-2-N genes. This study can provide a reference for future research on live vector vaccines for domestic animals, pets, and wild animals.

Seamless stitching of biosynthetic gene cluster containing type I polyketide synthases using Red/ET mediated recombination for construction of stably co-existing plasmids.

Type I polyketides are natural products with diverse functions that are important for medical and agricultural applications. Manipulation of large biosynthetic gene clusters containing type I polyketide synthases (PKS) for heterologous expression is difficult due to the existence of conservative sequences of PKS in multiple modules. Red/ET mediated recombination has permitted rapid manipulation of large fragments; however, it requires insertion of antibiotic selection marker in the cassette, raising the problem of interference of expression by leaving "scar" sequence. Here, we report a method for precise seamless stitching of large polyketide biosynthetic gene cluster using a 48.4kb fragment containing type I PKS involved in fostriecin biosynthesis as an example. rpsL counter-selection was used to assist seamless stitching of large fragments, where we have overcome both the size limitations and the restriction on endonuclease sites during the Red/ET recombination. The compatibility and stability of the co-existing vectors (p184 and pMT) which respectively accommodate 16kb and 32.4kb inserted fragments were demonstrated. The procedure described here is efficient for manipulation of large DNA fragments for heterologous expression.

Construction of the co-expression plasmids of fostriecin polyketide synthases and heterologous expression in Streptomyces.

CONTEXT: Polyketides are bioactive natural products with diverse bioactivities, and heterologous production of polyketides in easily engineered microbial hosts is preferred for the production of structurally diverse and the therapeutically active polyketides. OBJECTIVE: In this study, heterologous expression of the biosynthetic genes encoding type I polyketide synthases (PKS) involved in biosynthesis of fostriecin, a unique phosphate monoester polyketide antibiotic, was attempted. MATERIALS AND METHODS: Fostriecin PKS (Fos-PKS) biosynthetic gene cluster in a total of 48.4 kb were cloned downstream of the act I promoter in two compatible Streptomyces vectors using Red/ET recombination. The co-expression plasmids were sequentially transferred into Streptomyces lividans and Streptomyces coelicolor. Active transcription of the polyketide genes was confirmed by reverse transcription PCR (RT-PCR) analysis, and the metabolites were detected using high-performance liquid chromatography (HPLC). RESULTS: The recombinant strains S. lividans TK24/p6-fosAB-p4-fosCDEF and S. coelicolor M512/p6-fosAB-p4-fosCDEF were obtained for heterologous expression in Streptomyces. Pigmentation was observed in the recombinant strains, whereas the control strain with empty vector displayed no change in pigment production. Active transcription of the polyketide genes was confirmed by RT-PCR analysis and subsequent sequencing. CONCLUSION: The present study is the first attempt to overexpress Fos-PKS biosynthetic gene cluster in Streptomyces. More studies on heterologous expression of the fostriecin biosynthetic gene cluster would be beneficial for further understanding the mechanisms of its structural as well as the potential pharmaceutically effect.

Construction of Targeting Vector for Conditional Knockout of Murine Shbg / 中国医科大学学报

Objective To construct the targeting vector for conditional gene knockout of sex hormone?binding globulin(Shbg)in mice. Methods Based on Red/ET,two LoxP were inserted into both sides of extron 4 and extron 7 for conditional gene knockout of murine Shbg. Firstly,the Shbg gene and its upstream,downstream genes obtained from BAC DNA by PCR were cloned into plasmid pBR322?MK,which was named pBR322?MK?AB. The retrieve plasmid(pBR322?Shbg?Re)was obtained by homologous recombination between the plasmid and BAC.Then a great quantity of Neo fragments obtained from PL452 and PL451 were inserted into the targeting vector after another round of Red/ET and then the final targeting vec?tor(pBR322?MK?SHBG?cko)was achieved. Results The correct structures of the targeting vectors such as pBR322?MK?AB,pBR322?Shbg?Re, SHBG?Ln and pBR322?MK?Shbg?cko were confirmed by restriction enzyme digestion and sequencing analysis. Conclusion The targeting vector for conditional knockout of murine SHBG was successfully constructed. The construction of targeting vector paved the way for conditional knockout mouse strain generated by targeted mutation of Shbg.

Red/ET recombination with chimeric oligonucleotides allows rapid generation of BAC transgenes harboring full-length or truncated huntingtin cDNA.

Huntington's disease (HD) is a fatal neurodegenerative disorder that is caused by a CAG repeat expansion encoding a polyglutamine tract in the huntingtin (htt) gene. None of the existing HD mouse models recapitulate the exact disease symptoms and course as it is seen in humans and the generation of further HD disease models is challenging because of the size and complexity of the htt gene locus. Starting from a single substrate plasmid harboring human htt cDNA comprising 98 glutamine (Q) residues, we applied Red/ET recombination to generate four BDNF-BAC transgenes harboring full-length or truncated (N171) htt cDNA comprising 98 or 15 Q residues. BDNF (brain-derived neurotrophic factor) is expressed in the cortical neurons projecting to the striatal medium spiny neurons, and was used to direct htt transgene expression to investigate the contribution of these cell types to HD.