Expression Of Cytadheresin GapA And Construction Targeted Gene Vectors For Mycoplasma Gallisepticum

Mycoplasma gallisepticum (MG) is an important pathogen of poultry, causing chronic respiratory disease in chickens and infectious sinusitis in turkeys. Mycoplasma gallisepticum infection has a high prevalence throughout the world with significant economic impacts on the poultry industries, and renders birds more susceptible to stress and other infectious agents, eg: Infectious Bronchitis virus (IBV), Newcastle disease virus (NDV), etc.Attachment of MG to specific target cells via sialic acid residues along the respiratory epithelium is required prior to initiation of the disease processes. Therefore, in this study, the primary cytadhesin, GapA, was chosen to research pathogenesis of MG and targeted gene vectors were constructed as transposon vector, mini-Tn4001tetM, and a replicable vector based on MG oriC region.1 Comparison of gapA gene in virulent and avirulent strains of MGThe aims of this study were to amplify the 5'terminal hypervariable region of gapA gene in difference virulence strains by RT-PCR and PCR analysis. Compared of variability at a transcriptional level, or a translational one, the results indicated gapA gene residues in a large-size mRNA transcript. In the mRNA fragment, licA, mgc2, gapA and crmA are made up a virulent-related gene cluster. The 5'terminus of gapA gene exists a significant difference, in Rhigh strain, non-sense mutation is caused by a ORF shift by inserting an additional"A"residue at several sites 105nt, 386nt, and 912nt.In ts-11 strain, a point-mutation appeared at 142nt varied from"C"to"A", which led to a stop coden at the same place. However, in virulent strains (Rlow, S6, and DC9604) and two vaccine strains (F and 6/85), a complete GapA protein is encoded with a weight mass of about 110~120kD. Sequencing the gapA genes, we found that significant difference varies in the N terminus from 100aa to 300aa.2 High expression of N terminus of GapA cytadhesin (GapAN) in Rlow strainN terminus of GapA polypeptide from 98 to 322aa (GapAN), and C terminus polypeptide from 882aa to 1115aa (GapAC) were amplified. The fragments were subcloned pGEX-6P-1 vector, transformed into BL21 (DE3) competent cells, respectively. Induced by IPTG, the bacteria were identified by SDS-PAGE analysis. The results indicated GapAN was highly expressed in inclusive body. However, GapAC couldn't be successfully expressed. Meanwhile, gapAN gene was subcloned into pFAST-Bac1 vector. The following procedure referred to the handbook of Bac-to-Bac Baculovirus Expression System. A recombined Baculovirus was designated as rGapAN, identified with a polysera against GapAN by Indirect Immunofluorescence assay (IFA).3 Preparation of monoclonal antibodies against GapANGapAN expressed by prokaryotic system was separated, and the positive band was excised and immunized Balb/c mice. After 4 times, the spleen cells were fused with SP2/0 cells to parpare monoclonal antibodies. The Sf9 infected with rGapAN virus were used as a selective agent. The results showed that five hybridoma cell lines were successfully obtained and designated as 1B5, 1D7, 3D4, 4B3, and 5H10, respectively. The McAbs couldn't react with MG by HI and SPA analysis. 1B5, 3D4, 4B3 and 5B10 were highly specific to GapA by Western-blotting analysis except for 1D7, showing a specific band of about 120kD. However, 1D7 could react only with the purified GapAN antigen and product of Bac-to-bac expression system rather than the GapA protein expressed in M. gallisepticum. What's interesting, 5B10 could react with Rlow, DC9604, and S6 virulent strains rather than avirulent strains, such as ts-11, 6/85, and F. It might recognize with a high variant domain of GapAN, suggesting 5B10 could be used as a tool for identification of MG virulent strains.4 Construction of a transposon vector of mini-Tn4001tetM in MGIn order to study on functions of cytadhesin GapA, in this study, a kind of transposon vector was constructed and designated as mini-Tn4001tetM. In accordance with the principle of Tn4001 transposon, the transposon vector, mini-Tn4001tetM, contains tnp gene encoding a transposase gene in Staphylococcus aureus and two copies of IS256 in inverted repeat sequence (Inner and outer), conferring resistance gene to tetracycline, tetM, from Enterococcus faecalis Tn916 transposon. Further study on promotion of the vector, replacement of lacZ gene and tuf promoter was made to reconstitute screening genes and promoters of interest genes. The vector was then electro-transformated into MG and the recombinant cells were screened by Tetracycline. The original results indicated that the transposon vector could residue in MG strain R by successive passages. The further identification work on GapA or other function agents will be carried out.5 Construction of a replicable vector based on the oriC in MGAccording to the putative origin of replication (oriC) of MG strain Rlow, different primers were designed to amply several oriC regions. According to the size of base-pairs, the fragments were designated as long oriC (LoriC), short oriC (SoriC) and whole oriC (WoriC). And then the fragments were inserted into pBT vector (pBlueScript SK (+) containing tetM resistance gene from Tn916). The recombinant flanks will be subcloned to the replicable vector according to selection of interest genes.