| BackgroundGonorrhea is at the present time one of the most widespread venereal disease. The causative agent of the disease is the Neisseria gonorrhoeae, a bacterium, which can not only cause purulent infection of urogenital tract, but also invade to eyes, throat, and rectum. It sometimes causes systemic infections. Since Neisseria gonorrhoeae has throughout its history developed resistance to traditional antibiotics, it is very important to develop an effective vaccine to prevent the infection of Neisseria gonorrhoeae.The outer membrane of Neisseria gonorrhoeae includes a variety of proteins, the most abundant of which is known as protein I (PI), occupying about 60% of total outer membrane protein. PI may function as a porin creating a hydrophilic diffusion channel and allow uptake of essential nutrients. Unlike the other major surface antigens, pili, PII and LPS, PI dose not undergo antigenic variation during infection. PI may also play an important role in pathogenesis through its ability to interact with the cell membrane of eukaryotic cells. PI is also a major target for immune attack on the gonococcusand can stimulate the production of opsonins. And anti-Pi antibody can also activate complement-mediated bactericidal activity. Thus, by virtue of its stability and antigenicity, PI is of considerable interest as a potential gonococcal vaccine candidate.The goal of our study is to clone and express the gonococcal outer-membrane PI protein, and it may lead to a potential possibility for the further study of immune functions of PI.MethodsConstruction of recombinant:l,Polymerase chain reaction (PCR):Gene of recombinant gonococcal PI protein was amplified by PCR . Upper primer 5'-GGA TCC ATG AAA AAA TCC CTG ATT GCC CTG-3'; reverse primer 5'-CTT AAG CTT AAA CAC CGC GTC TGG CTG TGG -3'.The products of PCR reaction must be analyzed by agarose gel electrophoresis, and be purified by glass milk kit.2,Ligation and transformation:PCR product was inserted into T-Easy-Vector to form clone recombinant (T-Easy-Vector/Pi). After authenticating the DNA sequence, gene of PI protein was inserted into pGEX-4T-2 to form expression recombinant (pGEX-4T-2/PI).Induction and purification of GST-PI fusion protein:GST-PI fusion protein was induced according to the manual of GST Gene Fusion System. The purification process was: SDS-PAGE, excision of 60 000 protein band, and electroelution to collect GST-PI fusion protein.Authentication of recombinant PI protein:A dot immunochromatographic assay was employed to authenticate our purified GST-PI fusion protein. A purple band develops which indicates that our purified GST-PI fusion protein is gonococcal PI protein specific.Test of adhesion inhibition:Gonococcal colonies from clinical patients were suspended in 1ml DMEM, 0.5ml or 0.25ml fresh rabbit antiserum in DMEM respectively. The gonococci and rabbit antiserum were first incubated for 30min at 3 7 癈 with 6% COj. Then cocultures of HeLa cells and gonococci were incubated at 37癈 with 6% CO2 for 3hrs.Removed the fluid, monolayers were washed twice with antibiotics free DMEM. Observe the results under oil lens of microscope after gram stain.Results^Authentication of cloned PI gene fragments:Our cloned gene of gonococcal PI protein was a 1kb oligonucleotide. Its DNA sequence was the same as GI " 3925491 " [GenBank] Neisseria gonorrhoeae on PubMed.2,The induction, solubility analysis and purification of GST-PIfusion protein:We put the induced and non-induced E.coli containing pGEX-4T-2/PI undergoes SDS-PAGE analysis. Compared with the non-induced E.coli, the induced E.coli was demonstrated a deep stained new band at molecular weight of 60 000. The result suggested that the actual molecular weight of our induced GST-PI fusion protein should be about 60 000. As we know ,the molecular weight of GST protein and PI protein according to articles are 27 000 and 33000, so the calculated molecular weight of GST-PI fusion protein is also 60 000. Therefore, we got spec... |
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