Analysis of endogenous plasmids in Lacticaseibacillus paracasei ZY-1 and development of expression vectors / 生物工程学报

The construction of efficient and stable Lactobacillus expression vector is critical for strain improvement and development of customized strains. In this study, four endogenous plasmids were isolated from Lacticaseibacillus paracasei ZY-1 and subjected to functional analysis. The Escherichia coli-Lactobacillus shuttle vectors pLPZ3N and pLPZ4N were constructed by combining the replicon rep from pLPZ3 or pLPZ4, the chloramphenicol acetyltransferase gene cat from pNZ5319 and the replicon ori from pUC19. Moreover, the expression vectors pLPZ3E and pLPZ4E with the promoter Pldh3 of lactic acid dehydrogenase and the mCherry red fluorescent protein as a reporter gene were obtained. The size of pLPZ3 and pLPZ4 were 6 289 bp and 5 087 bp, respectively, and its GC content, 40.94% and 39.51%, were similar. Both shuttle vectors were successfully transformed into Lacticaseibacillus, and the transformation efficiency of pLPZ4N (5.23×102-8.93×102 CFU/μg) was slightly higher than that of pLPZ3N. Furthermore, the mCherry fluorescent protein was successfully expressed after transforming the expression plasmids pLPZ3E and pLPZ4E into L. paracasei S-NB. The β-galactosidase activity of the recombinant strain obtained from the plasmid pLPZ4E-lacG constructed with Pldh3 as promoter was higher than that of the wild-type strain. The construction of shuttle vectors and expression vectors provide novel molecular tools for the genetic engineering of Lacticaseibacillus strains.

In situ real-time evaluation of radiation-responsive promoters in the extremely radioresistant microbe Deinococcus radiodurans.

A third generation promoter probe shuttle vector pKG was constructed, using the green fluorescent protein as a reporter, for in situ evaluation of Deinococcal promoter activity in Escherichia coli or Deinococcus radiodurans. The construct yielded zero background fluorescence in both the organisms, in the absence of promoter sequences. Fifteen Deinococcal promoters, either harbouring Radiation and Desiccation Response Motif (RDRM) or not, were cloned in vector pKG. Only the RDRM-promoter constructs displayed (i) gamma radiation inducible GFP expression in D. radiodurans, following gamma irradiation, (ii) DdrO-mediated repression of GFP expression in heterologous E. coli, or (iii) abolition in GFP induction following gamma irradiation, in pprI mutant of D. radiodurans. Utility of pKG vector for real-time in situ assessment of Deinococcal promoter function was, thus, successfully demonstrated.

Functional study of Treponema pallidum genes using Borrelia burgdorferi as a surrogate system / 中华微生物学和免疫学杂志

Objective To employ Borrelia burgdorferi( B. burgdorferi) , a culturable and genetical-ly transformable spirochete, as a surrogate system to study Treponema pallidum ( T. pallidum) gene function. Methods Bioinformatic analysis revealed that the T. pallidum gene tp0111 encodes the putative sigma factor RpoN. We constructed a B. burgdorferi shuttle vector harboring tp0111. The shuttle vector was then trans-formed into the B. burgdorferi rpoN mutant strain. The phenotype of the resulting B. burgdorferi strain was then determined. Results We successfully constructed the B. burgdorferi rpoN mutant carrying the T. palli-dum gene tp0111. We found that tp0111 could partially complement the B. burgdorferi rpoN mutant. Con-clusion This work provides the first experimental evidence showing that tp0111 is the rpoN gene of T. palli-dum. It also demonstrates that B. burgdorferi can be used as a surrogate system for studying T. pallidum gene function.

Expression of exogenous genes in Chlamydia muridarum by transfection with shuttle vectors / 中华临床感染病杂志

Objective To add an open reading frame in the shuttle vector of pGFP ∷ CM for transfection of exogenous genes into Chlamydia muridarum.Methods The sequence of plasmid pGFP ∷ CM and new open reading frame (including promoter of pgp4,mCherry gene of red fluorescence protein and transcription termination sequence of Chlamydia trachomatis CT579) were amplified by polymerase chain reaction (PCR),and the products were transfected into Stellar competent cells.The recombinant plasmids were identified by PCR,enzyme digestion and sequencing.Then the recombinant plasmid was transfected into plasmid-free strain CMUT3,and the GFP-and mCherry-positive inclusions were observed under the fluorescence microscope.After the ampicillin selection and plaque purification,the purified CMUT3-pGFP-mCherry-CM was identified by indirect immunofluorecesent stain using anti-pgp3 and anti-glgA antibodies.Results The correct recombinant plasmid after sequencing identification,enzyme digestion and PCR amplification was successfully transfected into CMUT3,and the GFP-and mCherry-positive inclusions were observed.The transfected strain CMUT3-pGFP-mCherry-CM was purified after ampicillin selection and plaque purification.The expression of pgp3 and glgA protein in CMUT3-pGFP-mCherry-CM was similar to that in CMUT3-pGFP ∷ CM.Conclusion An open reading frame is successfully added in the plasmid pGFP ∷ CM,and the new plasmid can be transfected into CMUT3 and express exogenous protein,which can be used for further study on the function of single chlamydial protein.

Construction of a shuttle vector for inducible gene expression in Borrelia burgdorferi / 中华微生物学和免疫学杂志

Objective To construct a shuttle plasmid for inducible gene expression in Borrelia burgdorferi (B.burgdorferi) with an advantage of flexible genetic manipulation.Methods The IPTG-inducible lac repressor/operator system from Escherichia coli (E.coli) was adopted and modified in the current study.The plasmid shuttle vector was developed by inserting multiple cloning sites,FLAG and HA tags into the shuttle vector by molecular cloning approaches.The target gene was inserted at the site under the control of the promoter (Tn5 derivate) in plasmid pQE30.This promoter contained two lac operators and a codonoptimized lacI gene driven by flaB promoter.Results A plasmid shuttle vector,pJJ275,was successfully constructed with the ability to express target genes in B.burgdorferi in the presence of IPTG.By using this system,a HA-tagged rpoS gene was introduced into the typical infectious strain B.burgdorferi B31.The target gene expression induced by IPTG was confirmed at transcriptional and translational levels.The RpoS dependent virulence factor of Borrelia,OspC,was also detected,indicating that the expressed protein was functional.Conclusion The constructed plasmid shuttle vector can express exogenous genes in B.burgdorferi with an inducible feature and an advantage of flexible genetic manipulation.It can be applied for genetic manipulation of B.burgdorferi involved in gene regulation and complementation.

Construction of recombinant eukaryotic expression plasmid of human thyrotropin receptor extracellular domain encapsulated with cationic liposomes / 国际眼科杂志(Guoji Yanke Zazhi)

AlM:To construct recombination eukaryotic expression plasmid of human thyrotropin receptor extracellular domain encapsulated with cationic liposomes. METHODS:We amplified the target gene of shuttle vector PHMCMVTSHR289, conjugated the target gene and eukaryotic expression plasmid pcDNA3. 1 +, and accredited whether pcDNA3. 1+/TSHR289 was connected or not by enzymatic digestion and sequencing. Cationic liposomes encapsulated the recombination plasmid pcDNA3. 1+/TSHR289. RESULTS: Recombination plasmid pcDNA3. 1+/TSHR289 digested with enzyme Hindlll and the fragment through 0. 8% gel electrophoresis showed 512bp strip. Recombination plasmid pcDNA3. 1+/TSHR289 were found synonymous mutation through forward ( AAC to AAT ) and reverse sequencing ( GCG to GCT) . The volume ratio of cationic liposomes and recombinant plasmid was 3:1. CONCLUSlON: lt is successful to construct the recombination plasmid pcDNA3. 1+/TSHR289 by accredit it through enzymatic digestion and sequencing.

Expression of PDX1 in human umblical cord mesenchymal stem cells mediated by adenovirus vector / 中华内分泌外科杂志

ObjectiveTo construct recombinant adenovirus vector containing human pancreatic and duodenal homeobox factor 1 (PDX1) and detect its expression in human umblical cord mesenchymal stem cells (HUCMSCs). MethodsPDX1 obtained by BgⅢ/XhoI enzyme digestion from pUC57-PDX1 was ligated into the recombinant shuttle vector pShuttle-GFP-CMV to obtain the recombinant shuttle plasmid pShuttle-GFP-CMVPDX1. pShuttle-GFP-CMV- PDX1 was shifted to pAdxsi vector to obtain pAdxsi-GFP-PDX1 virus plasmid. The recombinant plasmid was packaged and amplified in 293 cells. The expression of PDX1 gene and protein in HUCMSCs was detected by fluorescence microscopy, RT-PCB, immunofluorescence, immunohistochemistry, and Western Blot. ResultsPDX1 gene was inserted correctly into shuttle plasmid and the recombinant adenovirus vector was successfully constructed according to the results of sequence and enzyme digestion identification. The adenovirus was effectively transfected into HUCMSCs. RT-PCR verified that PDX1 mRNA was positively expressed in HUCMSCs. Expression of PDX1 protein in the nuclear of HUCMSCs was found by immunofluorescence assay, immunohistochemistry and Western Blot. ConclusionThe adenovirus vector containing PDX1 gene is successfully constructed and effectively expressed in HUCMSCs.

Listeria seeligeri Gene Encoding Heme-containing Catalase Produces No Activity in Streptococcus pneumoniae

Streptococcus pneumoniae is a facultative anaerobe lacking catalase enzyme and requires exogenous catalase supplemented to culture media for aerobic growth. We introduced a catalase gene (kat) of Listeria seeligeri into S. pneumoniae and tried to see if this listerial kat gene was expressed within the pneumococcal host. To clone the listerial kat gene in the pneumococcal chromosome, a non-replicating plasmid pAHA-LSt3, along with its original promoter region was used for integration the chromosome via homologous recombination. One of three resulting transformants was confirmed to contain the kat gene and designated as EHS2. In addition, the kat gene was subcloned in Escherichia coli in frame to the lac promoter of a shuttle vector to generate pDL-Kat, which was subsequently used for pneumococcal transformation. Four identical recombinants were identified to contain the plasmid with the kat gene. By performing RT-PCR, it was observed that the listerial kat gene was indeed transcribed within pneumococcal recombinants from its original promoter in the chromosome of EHS2 and from the lac promoter in the plasmid pDL-Kat. In contrast to the E. coli kat+ recombinants, however, the pneumococcal kat+ recombinants failed to reveal any catalase activities detectable by ferricyanide staining on non-denaturing PAGE. When the pDL-Kat plasmid DNA purified from pneumococci was allowed to transform E. coli again, many kat+ recombinants were obtained, ruling out the possibility of the defective kat E. coli transformants gene within pneumococci. The observation that the listerial kat gene in pneumococci was unable to produce the functional catalase enzyme, which requires a heme group at its active site and a cofactor NADPH, suggests pneumococcal defect in heme production.

Factors affecting transformation efficiency of BCG with a Mycobacterium-Escherichia coli shuttle vector pYUB18 by electroporation

BCG has been one of the vehicles for multi-recombinant vaccine. However, low transformation efficiency of BCG with plasmid DNA hampered studies involving expression of foreign antigens in BCG. In an effort to determine the optimal conditions, this study was initiated to investigate factors involved in the transformation of BCG with a Mycobacterium-Escherichia coli shuttle vector, pYUB18, by electroporation. Mycobacterium bovis BCG (strain 1173P2) was grown in Middlebrook (M) 7H9 broth containing albumin-dextrose-catalase and 0.05% tween 80, and transformed BCG was grown in M7H10 agar containing kanamycin for counting viable cells. Pretreatment of BCG with 10 mM CaCl2 improved the transformation efficiency, but overnight incubation of BCG with 1% glycine did not. The transformation efficiency in BCG also varied depending on voltage, resistance, and DNA concentration. The maximum transformation efficiency was obtained when the infinity resistance, 12.5 Kv/cm, and 100 ng of DNA were used, and reached 1.4 x 10(5) CFU/microgram of plasmid DNA, which is about 3-100 times greater than those from previous reports. The transformation conditions described in this study, therefore, will give us a better position for employing BCG as a vehicle for developing multi-recombinant vaccines.

Construction of shuttle vectors for cloning in Vibrio cholerae and Escherichia coli.

Starting from a naturally occurring cryptic plasmid pVC540 of Vibrio cholerae non-OI. strain 1095, a number of plasmid vectors have been constructed for cloning genes in Vibrio cholerae by introducing antibiotic resistance markers containing a set of unique cloning sites. The constructs pVC810 and pVE920 have the origins of both Vibrio cholerae and Escherichia coli replicons and are stable in both organisms in the absence of selective pressure. These plasmids can serve as shuttle vectors between Escherichia coli and Vibrio cholerae. The plasmid vectors reported here along with the demonstration of transformation in Vibrio cholerae by plasmid DNA will facilitate genetic analysis of this important human pathogen.