本文選題：豬圓環(huán)病毒2型 + 全基因擴增��； 參考：《廣西大學(xué)》2015年碩士論文

【摘要】：豬圓環(huán)病毒2型(Porcine circo virus type 2, PCV2)于1998年被發(fā)現(xiàn)以來,現(xiàn)如今已成為一種廣泛流行于世界各地區(qū)的疾病病原之一。它嚴(yán)重危害豬群,破壞豬的淋巴系統(tǒng),使豬的淋巴細胞減少,進而造成豬的免疫力下降,處于亞健康狀態(tài),容易受其他病原或者外界環(huán)境因素的影響而導(dǎo)致發(fā)病。因此,PCV2在全球范圍內(nèi)已造成了巨大經(jīng)濟損失并且還在制約著世界養(yǎng)豬行業(yè)的發(fā)展。為了解廣西PCV2的流行情況,本研究對2012年8月至2015年1月期間采集的廣西不同地區(qū)的284樣品淋巴結(jié)及肺臟組織,采用PCR方法進行PCV2檢測,共檢測出144份陽性樣品,陽性率達50.7%；其中春、夏、秋、冬四季的PCV2陽性率分別為42.4%(36/85)、91.5%(43/47)、38.7%(43/111)和53.7%(22/41)。說明了廣西豬群中普遍存在PCV2感染現(xiàn)象,并且在夏季流行較為嚴(yán)重。為進一步了解PCV2在廣西流行的主要基因型。本研究選取檢測為陽性的部分樣品進行PCV2全基因的克隆,成功克隆出45株P(guān)CV2全基因序列。遺傳進化分析結(jié)果發(fā)現(xiàn),其中6株屬于PCV2a型,39株屬于PCV2b型,其中14株屬于PCV2b-1A/B亞群,25株屬于PCV2b-1C亞群。說明了廣西部分地區(qū)PCV2b流行廣泛,其中又以PCV2b-1C亞群流行為主。結(jié)合本實驗所保存的24株P(guān)CV2全基因進行分析,充分說明了廣西PCV2亞群流行發(fā)展的變化規(guī)律,是由PCV2b-lA/B亞群向PCV2b-1C亞群轉(zhuǎn)變。本研究得到6株P(guān)CV2a與中國吉林疫苗株HM038034及匈牙利株AY256459共同處于PCV2a的分支上。所得14株P(guān)CV2b-lA/B與中國廣東株EF421971、中國安徽株GU450327、中國浙江株AY691169、中國河南株FJ440338、中國遼寧株HM776448及中國北京株EF524530處于同一分支上,而不處于中國浙江疫苗株AY686764、中國山西疫苗株HM641752和荷蘭株AF201897所在的分支上。本研究所得25株P(guān)CV2b-1C中,有23株與中國黑龍江株HM038017、中國湖北疫苗株FJ870971、中國上海疫苗株AY686763、中國湖北株AY291317及中國甘肅株FJ948168處于同一分支。另外2株P(guān)CV2b-1C與中國湖北株AY035820、印度尼西亞株EU302139和越南株JX506730及JX099782處于同一分支,并且為PCV2a與PCV2b-1C ORF2的重組株。45株P(guān)CV2全基因核苷酸的同源性達94.2-99.9%,45株P(guān)CV2各基因型之間ORF1核苷酸具有很高的同源性為96.5-100%,而ORF2的核苷酸同源性為88.9-99.9%�？梢钥闯鯫RF1較ORF2保守,PCV2三個亞型的主要差異表現(xiàn)在ORF2。45株P(guān)CV2 ORF1氨基酸同源性為97.5-100%之間,而45株P(guān)CV2 ORF2氨基酸同源性為87.2-100%之間。根據(jù)PCV2 ORF2推導(dǎo)出的氨基酸序列比對發(fā)現(xiàn),ORF2氨基酸序列中存在3個主要的變異度較高區(qū)域：53-91 aa、121-151 aa和185-215 aa,并且包含著3個Cap蛋白的抗原表位：69-83 aa、117-131 aa和193-207 aa, Cap蛋白主要的氨基酸差異位點也在其中。PCV2各基因型主要在8、53、57、59、68、77、86-91、121、130、131、133、134、151、169、185、187、190-191、206、210、215、232、234的氨基酸位點上表現(xiàn)不同,D77、TNKISI86-91、P131、S133、I187、S190和K232是PCV2a區(qū)別于PCV2b的特點,F8、I53、N68、T121、N134、D215、K234是PCV2b中1C亞群區(qū)別于1A/B亞群的特點。為將廣西流行的PCV2毒株分離培養(yǎng)出來,本研究采用PK-15細胞帶毒傳代的方法,從144份PCV2陽性樣品中選取部分樣品在PK-15細胞中進行PCV2的分離,細胞帶毒傳代至10代時,采用PCR的鑒定方法,成功分離并鑒定出三株P(guān)CV2分離株GXGG-4-2013、GXNN-2-2014和GXCZ-2014。三株P(guān)CV2分離株同屬PCV2b型,其中GXGG-4-2013、 GXNN-2-2014屬于PCV2b-1C亞群,GXCZ-2014屬于PCV2b-1A/B亞群。GXGG-4-2013株經(jīng)過80代的帶毒傳代培養(yǎng),每隔十代擴增分離病毒的全基因,8個代次的全基因核苷酸序列同源性很高在99.5-100%之間,說明其核苷酸保持相對穩(wěn)定,未出現(xiàn)很大的變異。本研究分析廣西部分地區(qū)PCV2的流行情況及PCV2分子流行特點,進而為廣西PCV2的防控提供理論參考。分離出的3株P(guān)CV2可以為以后研究PCV2分離株的生物特性提供材料。
[Abstract]:Since its discovery in 1998, the porcine circovirus 2 (Porcine Circo virus type 2, PCV2) has now become one of the disease pathogens that are widely prevalent in all regions of the world. It seriously endangering pigs, destroying the lymphatic system of pigs, reducing the lymphocytes of pigs, and thus forming a pig's immune decline, being in a subhealthy state and easy to suffer from it. Therefore, PCV2 has caused huge economic losses worldwide and is still restricting the development of the world's pig industry. To understand the prevalence of PCV2 in Guangxi, 284 samples of lymph nodes and lungs in different regions of Guangxi collected from August 2012 to January 2015 A total of 144 positive samples were detected by PCV2 by PCR method, and the positive rate was 50.7%. The positive rates of PCV2 in spring, summer, autumn and winter were 42.4% (36/85), 91.5% (43/47), 38.7% (43/111) and 53.7% (22/41). It indicated that the prevalence of PCV2 infection in the Guangxi pigs was more serious in summer. To understand the main genotypes of PCV2 in Guangxi, this study selected the positive part of the sample to clone PCV2 whole gene and successfully cloned 45 PCV2 whole gene sequences. The results of genetic evolution analysis found that 6 of them belong to PCV2 A and 39 belong to PCV2 B type, of which 14 belong to PCV2b-1A/B subgroup and 25 belong to PCV2b-1C subgroup. It is clear that the prevalence of PCV2b is widespread in parts of Guangxi, among which PCV2b-1C subgroup is the main epidemic. Combined with the analysis of the 24 PCV2 whole genes preserved in this experiment, the variation of the epidemic development of the Guangxi PCV2 subgroup is fully explained, which is transformed from the PCV2b-lA/B subgroup to the PCV2b-1C subgroup. This study obtains 6 PCV2a and Jilin vaccine strain HM03803 in China. 4 and the Hungarian strain AY256459 are in the branch of PCV2a. The 14 strains of PCV2b-lA/B are on the same branch as China Guangdong strain EF421971, China Anhui strain GU450327, China Zhejiang strain AY691169, China Henan strain FJ440338, China Liaoning strain HM776448 and Beijing strain in China, but not in Zhejiang vaccine strain AY686764, China Shanxi. On the branch of the vaccine strain HM641752 and the Holland strain AF201897, 23 of the 25 PCV2b-1C strains obtained from this study were in the same branch of the Chinese Heilongjiang strain HM038017, the Hubei vaccine strain FJ870971 in China, the Shanghai vaccine strain of China, the Hubei strain AY291317 in China and the Gansu strain FJ948168 of China, and the other 2 strains of PCV2b-1C and Hubei strain AY035820 of China. The Indonesia strain EU302139 and the Vietnamese strain JX506730 and JX099782 are in the same branch, and the homology of the PCV2 full gene nucleotides of the recombinant strain.45 strain of PCV2a and PCV2b-1C ORF2 is 94.2-99.9%. The ORF1 nucleotide of the 45 PCV2 genotypes has a high homology for 96.5-100%. It is found that ORF1 is more conservative than ORF2, and the main difference between the three PCV2 subtypes is that the PCV2 ORF1 amino acid homology of the ORF2.45 strain is 97.5-100%, and the 45 PCV2 ORF2 amino acid homology is between 87.2-100%. According to the amino acid sequence alignment derived from PCV2 ORF2, there are 3 major regions of high variability in the amino acid sequence: 53-91 AA, 121-151 AA and 185-215 AA, and contain 3 Cap protein epitopes: 69-83 AA, 117-131 AA and 193-207 AA, and the main amino acid difference loci of Cap protein are also in the amino acid site of.PCV2, which are mainly in the amino acid site of 8,53,57,59,68,77,86-91121130131133134151169185187190-191206210215232234. D77, TNKISI86-91, P131, S133, I187, S190 and K232 are the characteristics that differ from PCV2b in PCV2a, F8, I53, N68, etc. When the samples were separated from the PK-15 cells for the separation of PCV2 and the cells were transmitted to the 10 generation, three PCV2 isolates, GXGG-4-2013, GXNN-2-2014 and GXCZ-2014., were isolated and identified by PCR. GXGG-4-2013 and GXNN-2-2014 belonged to the PCV2b-1C subgroup. After 80 generations of 4-2013 generations, the whole gene of the virus was amplified and separated every ten generations, and the nucleotide sequence of the 8 generations was highly homologous to 99.5-100%, indicating that the nucleotides remained relatively stable, and there was no large variation. This study analyzed the prevalence of PCV2 and the epidemic characteristics of PCV2 molecules in parts of Guangxi. It provides a theoretical reference for the prevention and control of PCV2 in Guangxi. 3 strains of PCV2 isolated can provide materials for further study of the biological characteristics of PCV2 isolates.
【學(xué)位授予單位】：廣西大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S858.28

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