Cloning, Prokaryotic Expression, And Function Of Glycoprotein G And The Recombination Of Infectious Laryngotracheitis Virus

The glycoprotein G (gG) gene and its flank sequences, including part of HSV-I UL47 homologue, complete gG gene and ORF5, of infectious laryngotracheitis virus (ILTV) of virulent Beijing E3 strain was amplified by PCR, cloned into pGEM-T vector and sequenced. The gG gene of ILTV vaccine strain Zhonghai was also amplified with another pair of primers and sequenced. Sequence analyses showed that the gG gene was very closed among sequenced ILTV strains. The deduced amino acids of most alphaherpesviruses' gG proteins were very conservative. They have a characteristic pattern of three closely-spaced cysteine residues. Other characteristics of gG protein were also analysed such as hydrophilicity, antigenic index, titration curve and the modification by phosphorylation sulfation glycosylation.The pMAL-c2x (E.coli BT1) and pET-30a(+) (E.coli BL21) prokaryotic expression system were used to express the gG excluding the signal sequence. The results showed that two systems showed high expression level. The pMAL-c2x (E.coli BT1) system expressed the soluble fusion protein MBP-GG was 25.8 percent of total bacterial protein and the Mr was about 72KDa of which the GG takes less 45 percent. The pET-30a(+) (E.coli BL21) system expressed the insoluble fusion protein His-GG was 18.3 of total bacterial protein and the Mr was about 34KDa of which the GG takes about 90 percent. After purification, those two kinds of fusion proteins were immunized the rats respectively to prepare their antibodies. Then the antibody was purified.The gG protein of the ILTV was purified from the allantoid fluid of the chicken's embryo by using the DEAE BIO-GEL. By adding the gG and its antibody to the culture medium of the primary CEL cell monolayers, the ILTV gG function was analyzed in ILTV attachment, penetration, direct cell-to-cell spread (CTCS) and the growth curve. The treatment with ILTV gG had no obvious affection to the virus infection, penetration, CTCS and growth curve. The attachment efficiencies of the E3 and Zhonghai were 85.6 and 79.1 percent respectively, and the penetration efficiencies of the E3 and Zhonghai were 68.9 and 62.9 percent respectively. The E3 and Zhonghai strains of the ILTV showed the characters of strong attachment and penetration. The gG antibody reduced the virus plaque size (50.96 percent (E3) and 56.73 percent (Zhonghai) of the control) and the one-step growth curve on CEL cells, but the gG treatment did not. There were several reasons for this result. One was that the ILTV gG was expressed at early and middle phase (data not shown) and was expressed strongly, which attenuated the function of the additive gG Little effect of the gG addition to the culture medium may also imply that the gG couldn't work by itself. Laser scanning confocal microscopy (LSCM) detection showed that the ILTV gG lied in perinuclear region and membrane of the CEK cells. Those results suggested that the ILTV gG might work in direct cell-to-cell transmission.The plasmids of pGEM-FgG/CMV-GFP, pGEM-FgG/SV40-GFP and pGEM-FgG/GFP expressing the green fluorescent protein (GFP) were constructed with the CMV, SV40 and the ILTV gG promoter as the promoter respectively and the SV40 polyA as the stop codes. By the homologous recombination method the expressing frames was inserted into the ILTV genome to replace the gG gene respectively. The recombinants of ILTV were detected by using fluorescent microscope. The stability of recombinants of ILTV was detected by a serial passage on chicken liver cells. The results showed that the plasmids, pGEM-FgG/CMV-GFP, pGEM-FgG/SV40-GFP and pGEM-FgG/GFP, could express the GFP and that the recombinant alive ILTV could be observed after three generations, the more generation we passed the less recombinant ILT virions that were observed and that the gG promoter need the ILTV protein(s) to express the gene. Those results suggested that the cotransfection of the lined plasmids pBL-ICP4, pGEM-FgG/CMV-GFP, pGEM-FgG/SV40-GFP and pGEM-FgG/GFP and the ILTV DNA genome could generate the recombinant alive ILT virions (ILTV ^-gG), but...