Study on development of HPV vaccine and the use for prevention of cervical cancer

1. 抗HPV L2的中和抗体能在体外交叉中和不同类型的HPV。为了鉴定含有HPV16 L2交叉中和表位的片段，我们用10个长14 ~ 20个氨基酸（aa）的合成肽分别免疫两只兔子获得了抗血清（AS），这些肽代表了HPV16 L2序列aa 14 ~ 144的一部分。AS针对结合HPV16 L1/ l2衣壳的肽和中和的HPV16假病毒粒子。抗肽aa 18 - 38（抗p18 /38）交叉中和HPV18。Anti-P56/75交叉中和HPV18、31和58。抗p61 /75和抗p64 /81交叉中和HPV18和58。抗P96/115和突变体P96/115交叉中和HPV31和hpv58诱导的AS。等体积的三种AS，抗p18 /38，抗p56 /75和抗突变体P96/115混合，比单独抗p56 /75更有效地中和HPV16, 18, 31和58。交叉中和似乎与更多HPV类型中的保守aa序列相关。这些数据为设计针对更广泛的高危hpv有效的疫苗抗原提供了基础。未分化细胞中hpv16ll的产生受到几个尚未确定的顺式作用抑制RNA元件（EL）的负调控，其中一个主要EL位于Ll- mrna的前514个核苷酸内。通过Northern blotting，我们检测了主EL对293T细胞中荧光素酶（Fluc）基因与编码主EL的Ll DNA片段联合瞬时转录的mRNA稳态水平的影响。EL下调mRNA的稳态水平。最有效的下调是通过在mRNA的5'端插入EL实现的。具有EL的mRNA的半衰期与正常的氟康mRNA相似。当通过剪接去除5'端附近的EL时，所得到的mRNA的稳态水平升高到易于检测的水平。这表明，编码主要抑制EL的DNA区域不干扰转录，在mRNA成熟过程中，在剪接步骤之后，pre-mRNA被RNA EL介导的机制降解。少

1. Neutralizing antibody against HPV L2 can cross-neutralize different HPV types in vitro. To identify the segments containing the cross-neutralization epitopes of HPV16 L2, we characterized antisera (AS) obtained by immunizing two rabbits with each of the ten synthetic peptides of 14 to 20 amino acids (aa) long, which represents a part of the HPV16 L2 sequence from aa 14 to 144. The AS against the peptides bound to HPV16 L1/L2-capsids and neutralized HPV16 pseudovirions. AS against the peptide from aa 18 to 38 (anti-P18/38) cross-neutralized HPV18. Anti-P56/75 cross-neutralized HPV18, 31, and 58. Anti-P61/75 and anti-P64/81 cross-neutralized HPV18 and 58. Anti-P96/115 and the AS induced by a mutant P96/115 cross-neutralized HPV31 and 58. The mixture of equal volumes of three AS, anti-P18/38, anti-P56/75, and anti-mutant P96/115, neutralized HPV16, 18, 31, and 58 more efficiently than anti-P56/75 alone. The cross-neutralization appears to be correlated with conserved aa sequences among … More HPV types. The data provide a basis for designing vaccine antigens effective against a broader spectrum of the high-risk HPVs.2. Production of HPV16 Ll in undifferentiated cells is negatively regulated by several yet unidentified cis-acting inhibitory RNA elements (ELs), among which a major EL is located within the first 514 nucleotides of the Ll-mRNA. By Northern blotting we examined effect of the major EL on the steady-state level of mRNA transiently transcribed in 293T cells from the firefly luciferase (Fluc) gene combined with the Ll DNA fragment encoding the major EL. The EL down-regulated steady-state level of the mRNA. The most efficient down-regulation was achieved by insertion of the EL near the 5' end of mRNA. The half-life of the mRNA having the EL was similar to that of normal Fluc-mRNA. When the EL near the 5' end was removed by splicing, the steady-state level of the resultant mRNA was raised to a readily detectable level. It is suggested that DNA region encoding the major inhibitory EL does not disturb transcription and that the pre-mRNA is degraded by an RNA EL-mediated mechanism after the splicing step in the course of mRNA maturation. Less