江西猪群中1型和2型猪细环病毒流行病学调查及全基因扩增、序列分析

发布时间：2018-06-04 01:02

本文选题：猪细环病毒1型和2型 + 感染情况　；参考：《江西农业大学》2015年硕士论文

【摘要】：猪细环病毒(Torque teno sus virus,TTSuV)是一种无囊膜、环状、单股负链DNA病毒,病毒粒子直径约30~32 nm。人源TTV DNA病毒是由日本学者Nishizawa等在1997年使用代表性差异分析技术发现的一种新病毒。1999年美国学者首次从猪血清中分离到,随后不断有学者相继在多种家养及野生动物体内检测出细环病毒。猪细环病毒作为一种小DNA病毒,在世界范围内猪群中被广泛检出,且该病毒会直接或间接的影响猪群的健康,带来经济损失,引起了养猪业极大的关注。本文采用已建立的套式PCR法检测了自江西8个地区不同猪场采集的血液样品,调查了猪细环病毒1型和2型的感染情况。然后,分别使用一对特异性扩增1型和2型TTSuV全基因组序列,并分析了全基因序列及主要功能基因的遗传进化关系。收集的183份血液样品进行了猪细环病毒1型和2型的检测,检测结果显示猪细环病毒1型的感染率为40.4%,猪细环病毒2型的感染率为68.9%,其中猪细环病毒1型和2型混合感染数共55份,占36.1%。参考GenBank公布TTSu V1和TTSuV2全基因序列,参考毒株AB076001和AY823991分别设计了1对扩增TTSuV1和TTSuV2全基因扩增特异性引物,使用长片段扩增酶得到猪细环病毒全基因序列。通过T-A克隆、序列测定与拼接获得了10株TTSuV的全基因组序列,其中6株TTSuV1毒株基因组全长分别为2879 bp、2913 bp、2860 bp、2909 bp、2894 bp、2906 bp,4株TTSuV2毒株基因组全长分别为2800 bp、2796 bp、2795 bp、2793 bp。通过对试验得到的TTSuV全基因序列的同源性与遗传进化分析发现:试验得到的6株TTSuV1全基因组序列与GenBank已有全基因组序列的相似性均在75%~97%,4株TTSuV2全基因组序列的相似性均在76%~98%;系统进化树分析表明,TTSuV1-JX2与北京HM633243毒株亲缘关系最近,TTSuV1-JX3与德国GU188045毒株亲缘关系最近,TTSuV1-JX4与西班牙GU570198毒株亲缘关系最近,而TTSuV1-JX5、TTSuV1-JX6两株独立成一个分支。TTSuV2-JX1与西班牙GU570206毒株亲缘关系最近,TTSuV2-JX2、TTSuV2-JX3、TTSuV2-JX4都与西班牙GU570207毒株亲缘关系最近。通过对TTSuV1-JX3和TTSuV2-JX3各1株全基因组的ORF1蛋白分析发现,两株TTSuV的ORF1分别包含639个和623个氨基酸,TTSuV1-JX3的ORF1在8~39、44~63、88~115、307~322、590~627等位点处的氨基酸抗原表位相对最强,TTSuV2-JX3 ORF1蛋白在9~49、179~202、281~302、570~581、590~609等位点处的氨基酸抗原表位最强。
[Abstract]:Porcine teno sus virus (TTSuv) is a membrane-free, annular, single-stranded DNA virus with a diameter of 30 ~ 32 nm. Human TTV DNA virus is a new virus discovered by Japanese scholar Nishizawa et al in 1997 using representative differential analysis technique. In 1999, American scholars first isolated the virus from pig serum. Subsequently, scholars have been detected in a variety of domestic and wildlife fine loop virus. As a small DNA virus, porcine fine loop virus is widely detected in swine herd worldwide, and the virus will directly or indirectly affect the health of pig herd and bring economic loss, which has aroused great concern in pig industry. The blood samples collected from different pig farms in 8 areas of Jiangxi Province were detected by nested PCR method. The infection of porcine fine loop virus type 1 and type 2 was investigated. Then, the whole genome sequences of type 1 and type 2 TTSuV were amplified by a pair of specific amplification methods, and the genetic evolution of the whole gene and the major functional genes were analyzed. A total of 183 blood samples were collected for the detection of porcine fine loop virus type 1 and type 2. The results showed that the infection rate of porcine fine loop virus type 1 was 40.4 and the infection rate of porcine fine loop virus type 2 was 68.9. The number of mixed infection of porcine fine loop virus type 1 and type 2 was 55 (36.1%). The whole gene sequence of TTSu V1 and TTSuV2 was published by referring to GenBank, and a pair of primers for amplification of TTSuV1 and TTSuV2 were designed by referring to AB076001 and AY823991, respectively. The whole gene sequence of porcine fine circovirus was obtained by using long fragment amplification enzyme. Through T-A cloning, the whole genome sequences of 10 TTSuV strains were sequenced and sequenced. The total genome length of 6 TTSuV1 strains was 2879 BP, 2913 BP, 2860 BP, 2909 BP, 2894 BP, 2906 BP, and the genome length of 4 TTSuV2 strains was 2800 BP, 2796 BP, 2795 BP, 2793 BP, respectively. By analyzing the homology and genetic evolution of the whole TTSuV gene sequence obtained from the experiment, it is found that the similarity of the 6 TTSuV1 genome sequences obtained from the experiment with the existing GenBank genome sequences is similar to that of the whole TTSuV2 genome sequence of the four strains of TTSuV2. Phylogenetic tree analysis showed that TTSuV1-JX2 was closely related to Beijing HM633243 strain. TTSuV1-JX3 had the closest genetic relationship with GU188045 strain in Germany, and TTSuV1-JX4 had the closest relationship with GU570198 strain in Spain. The relationship between TTSuV2-JX1, TTSuV2-JX1, TTSuV2-JX2, TTSuV2-JX2, TTSuV2-JX3, TTSuV2-JX3, TTSuV2-JX4, and Spanish GU570207 strain, was the most close relationship between TTSuV1-JX5, TTSuV1-JX6, TTSuV2-JX1, TTSuV2-JX1, TTSuV2-JX2, TTSuV2-JX2 and TTSuV2-JX2-JX4. Through the analysis of the whole genome of TTSuV1-JX3 and TTSuV2-JX3, it was found that, The ORF1 of two strains of TTSuV contained 639 amino acids and 623 amino acids, respectively. The amino acid epitopes of TTSuV1-JX3 were the strongest at the allelic point of 8H39An 446368838807 and 322590C627. The amino acid epitopes of TTSuV2-JX3 ORF1 protein were the strongest at the allele point 9491717282828130257070581590909. The amino acid epitopes of the two strains were the strongest at the allelic point of 949172821, 3025707, 58159090609.
【学位授予单位】：江西农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.28

【相似文献】