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Preliminary Studies on the Establishment of Transgenic Mice Expressing Human CD46 and CEACAM1 Receptors for Neisseria Gonorrhoeae Adherence Factors

Author: WangJinSong
Tutor: SunHuaiChang;LiGuoCai
School: Yangzhou University
Course: Zoology
Keywords: Neisseria gonorrheae adherence factors hCD46 hCEACAM1 transgenic mice
CLC: Q78
Type: Master's thesis
Year: 2007
Downloads: 30
Quote: 0
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Abstract


Neisseria gonorrhoeae (also called gonococcus), a species of Gram-negative diplococcus, is the causative agent of the sexually transmitted disease (STD) gonorrhea which mainly causes acute or chronic purulent infection on the mucous membrane of the urogenital system. Gonorrhea is one of the most prevalent STD, with 62 million reported cases annually worldwide. The treatment of gonorrhea is increasingly problematic due to the high frequency of acquisition of resistance to multiple antibiotics. It will be key to develop alternative treatments and effective vaccines in the control of gonococcal infection. However, studies on gonorrhea are challenged by the lack of useful animal models for gonococcal diseases because of the susceptibility of N. gonorrhoeae restrictive to human. It is reported that there are some specific receptors expressing in human for N. gonorrhoeae infection, e.g., hCD46 and hCEACAM1 are the receptors for pili and Opa proteins, respectively. Therefore, we hypothesize that transgenic mice expressing hCD46 and hCEACAM1 is a useful animal model for gonorrhoea research.To establish the transgenic mice that mimic the expression properties of human CD46, the promoter region of human CD46 gene was cloned by PCR using genomic DNA of HeLa cells as template. Sequence analysis demonstrated that the PCR amplified hCD46 gene promoter was only 96.4% homologous to that published in 1993, differing by 43 nucleotides in 21 locus, and was 99.9% homologous to a DNA fragment in the 5’end of hCD46 gene published in 2006, differing by only 2 nucleotides. The cloned promoter was used to direct the expression of enhanced green fluorescence protein (EGFP), and the rabbitβ-globin intron 2 was used to enhance the expression level. It was showed that the expression level of EGFP in CHO cells was much higher than that in SP2/0 cells, being similar to the CD46 expression properties in vivo in human. The rabbitβ-globin intron 2 enhanced the EGFP expression markedly in both of the 2 cell lines without affecting the tissue specificity of the hCD46 promoter. Hence, the cloned hCD46 promoter keeps its original promoting characteristics.As the CD46 promoter is conterminous with the extron 1 in human genome, a fusion hCD46 minigene containing the promoter and the cDNA was amplified by ligation-mediated PCR and subcloned into pcDNA3.1 replacing the original CMV promoter. The resultant recombinant eukaryotic expression vector was named pCD46. Another recombinant vector pCDPCAM1-GI, containing hCD46 promoter, rabbitβ-globin intron 2 and hCEACAM1 cDNA, was constructed. COS-1 cells were transfected with pCD46, pCDPCAM1-GI and pCD46 + pCDPCAM1-GI, selected by G418, respectively. Expression of hCD46 or/and hCEACAM1 was revealed by FACS using hCD46 or hCEACAMs specific McAbs. N. gonorrheae could bind to all the 3 transgenic cell lines, but not the non-transgenic COS-1 cells, and levels of bacterial adherence were highest in co-transgenic cell lines. These data indicate that the constructs, pCD46 and pCDPCAM1-GI, are useful for development of transgenic animal models for studying gonococcal diseases.Mice zygotes were microinjected with hCD46, hCEACAM1 and hCD46 + hCEACAM1 gene construction, and 48, 22 and 49 F0 mice were obtained, respectively. 4 (1♀3♂) were showed to be hCD46 transgenic by PCR. The expression of integrated interest gene in hCD46 transgenic mice, as well as the intergration and expression of hCEACAM1 and hCD46 + hCEACAM1 gene in relative F0 transgenic mice, are still to be analyzed further.

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