【摘要】： 目的:了解黑龍江省肇東市自然人群中乙型肝炎病毒(HBV)基因型、血清亞型分布情況及基因型與血清亞型的關(guān)系;了解自然人群中HBV S基因的核苷酸水平變異及氨基酸水平變異情況;分析HBV基因型與臨床轉(zhuǎn)歸之間的關(guān)系。 方法:2005年,以黑龍江省肇東市雙勝村和先鋒村兩個(gè)農(nóng)村為研究現(xiàn)場(chǎng),以全屯居民為研究對(duì)象,對(duì)全屯居民進(jìn)行了一次橫斷面血清流行病學(xué)調(diào)查,采用固相放射免疫(SPRIA)法檢測(cè)HBsAg、抗-HBs和抗-HBc三項(xiàng)乙肝病毒感染指標(biāo),采用酶聯(lián)免疫(ELISA)法檢測(cè)HBeAg和抗-HBe兩項(xiàng)感染指標(biāo)。結(jié)合1986年這兩個(gè)現(xiàn)場(chǎng)的血清流行病學(xué)調(diào)查資料,綜合ALT、B超和臨床體檢等結(jié)果,按照2005年12月10日制定的《慢性乙型肝炎防治指南》的診斷標(biāo)準(zhǔn),選擇可作出明確臨床診斷的110例作為研究對(duì)象。其中無(wú)癥狀HBV攜帶者(ASC)86例,慢性乙型肝炎(CH)12例,肝硬化(LC)5例,肝細(xì)胞肝癌(HCC)1例,恢復(fù)6例。男性60例,女性50例。平均年齡36歲。對(duì)從110例研究對(duì)象中抽取的血清標(biāo)本采用巢式PCR法擴(kuò)增HBV DNA。利用QIAamp DNA Blood Mini Kit,按照試劑盒操作說(shuō)明書(shū)要求從200μl血清中提取HBV DNA,將其作為模板,設(shè)計(jì)合成2對(duì)引物擴(kuò)增HBV S基因。擴(kuò)增產(chǎn)物經(jīng)1%瓊脂糖凝膠電泳鑒定后,應(yīng)用Wizard SV Gel and PCR Clean-Up System試劑盒進(jìn)行純化回收。將回收后的產(chǎn)物送生物公司利用ABI 3773自動(dòng)熒光測(cè)序儀測(cè)序。利用DNASTAR軟件對(duì)測(cè)序標(biāo)本的核苷酸序列與從GenBank中獲取的HBV A-H各基因型標(biāo)準(zhǔn)序列進(jìn)行比對(duì)分析,繪制基因進(jìn)化樹(shù),確定基因型。由核苷酸序列推導(dǎo)氨基酸序列,確定血清亞型。應(yīng)用SAS 8.1軟件對(duì)實(shí)驗(yàn)結(jié)果進(jìn)行Fisher確切概率法的統(tǒng)計(jì)學(xué)處理分析,以P0.05為有統(tǒng)計(jì)學(xué)意義。 結(jié)果: 110例血清標(biāo)本中,97例HBV DNA陽(yáng)性,陽(yáng)性率為88.2%。97例標(biāo)本的基因型分布為:B基因型9例,占9.3%;C型87例,占89.7%;D型1例,占1.0%。血清亞型分布為:adr亞型77例,占79.4%;adw2亞型11例,占11.3%;ayr亞型8例,占8.2%;ayw2亞型1例,占1.0%。9例B基因型標(biāo)本的血清亞型均為adw2亞型;87例C基因型標(biāo)本的血清亞型為: adrq+ 75例(占86.2%),adrq- 2例(占2.3%),adw2 2例(占2.3%);ayr 8例(占9.2%);1例D基因型標(biāo)本的血清亞型為ayw2亞型。用Fisher確切概率法對(duì)不同基因型的血清亞型構(gòu)成進(jìn)行統(tǒng)計(jì)分析,P值為0.0000,有統(tǒng)計(jì)學(xué)意義。 9例B基因型感染者中,4例(44.4%)為ASC,5例為CH(55.6%);87例C基因型感染者中,3例為恢復(fù)者(3.4%),73例為ASC(83.9%),7例為CH(8.1%),3例為L(zhǎng)C(3.4%)和1例為HCC(1.2%);1例D基因型感染者為ASC(100%)。Fisher確切概率法對(duì)不同基因型的臨床轉(zhuǎn)歸進(jìn)行統(tǒng)計(jì)分析,P值為0.0288,有統(tǒng)計(jì)學(xué)意義。 本次試驗(yàn)共獲得84例HBV S基因540nt完整序列結(jié)果。通過(guò)對(duì)這些核苷酸序列的堿基替換位點(diǎn)以及由核苷酸序列推導(dǎo)出的氨基酸序列的突變位點(diǎn)分析,發(fā)現(xiàn)131個(gè)位點(diǎn)有堿基替換現(xiàn)象,其中40個(gè)位點(diǎn)為同義突變, 89個(gè)位點(diǎn)為錯(cuò)義突變,2個(gè)位點(diǎn)為無(wú)義突變。主要結(jié)構(gòu)同源區(qū)(MHR,aa100-160)發(fā)現(xiàn)45個(gè)位點(diǎn)有堿基替換現(xiàn)象,其中18個(gè)位點(diǎn)為同義突變,27個(gè)位點(diǎn)為錯(cuò)義突變。錯(cuò)義突變中有2個(gè)位點(diǎn)產(chǎn)生了有意義的氨基酸突變—Q129H(1例)和M133L(1例),位于抗原決定簇a的第一個(gè)莖環(huán)結(jié)構(gòu)內(nèi)。1例在aa119~120間有三個(gè)氨基酸TTE的插入。HBV S基因每100個(gè)核苷酸替換的頻數(shù)為2.19。 結(jié)論: 1黑龍江肇東市HBV基因型分布以C基因型為主,存在少量的B型和D型。 2黑龍江肇東市HBV血清亞型分布以adr亞型為主(adrq+和adrq-均有,但以adrq+為主),存在少量的adw2、ayr和ayw2亞型。 3 HBV不同基因型的血清亞型的構(gòu)成不同。B基因型標(biāo)本的血清亞型均為adw2;C型標(biāo)本的血清亞型主要為adr,有少量的adw2和ayr;D型標(biāo)本的血清亞型為ayw2。提示HBV基因型與血清亞型存在一定的相關(guān)性。 4 HBV不同基因型的臨床轉(zhuǎn)歸不同。B基因型感染者中ASC和CH均有,CH與ASC者相比稍多。C基因型感染者中以ASC為主,CH次之,其次為恢復(fù)、LC和HCC。提示B基因型感染者較易形成CH,C基因型感染者各種臨床轉(zhuǎn)歸均有,絕大多數(shù)為ASC。 5 HBV S基因在許多位點(diǎn)發(fā)現(xiàn)堿基替換現(xiàn)象,但近1/3堿基替換為同義突變。自然人群中存在一定的HBV S基因突變,未發(fā)現(xiàn)疫苗相關(guān)突變。核苷酸替換的平均頻數(shù)較低,提示S基因相當(dāng)保守。
[Abstract]:Objective: to understand the genotype of hepatitis B virus (HBV), the distribution of serum subtypes and the relationship between the genotype and the subtype of serum in the natural population of Zhaodong, Heilongjiang, and to understand the variation of the nucleotide level and the amino acid level of the HBV S gene in the natural population, and to analyze the relationship between the HBV type and the clinical outcome.
Methods: in 2005, two rural areas of Zhaodong city and Xianfeng village in Zhaodong, Heilongjiang Province, were used as the research sites. The residents of whole Tuen Tuen were studied. A cross-sectional sero epidemiological survey was carried out on the residents of Tuen Tuen. The solid phase radioimmunoassay (SPRIA) was used to detect the HBsAg, anti -HBs and anti -HBc hepatitis B virus infection indexes, and the enzyme linked immunization (ELI) was used. SA) was used to detect two indexes of HBeAg and anti -HBe infection. Combined with the data of the sero epidemiological investigation of the two sites in 1986, combined with the results of ALT, B ultrasound and clinical examination, according to the diagnostic criteria of the guide for the prevention and treatment of chronic hepatitis B made in December 10, 2005, 110 cases of clinical diagnosis were selected as the research object. 86 cases of symptomatic HBV carriers (ASC), 12 cases of chronic hepatitis B (CH), 5 cases of cirrhosis (LC), 1 cases of hepatocellular carcinoma (HCC), 6 cases, 60 males and 50 females. The average age was 36 years old. The serum samples extracted from 110 subjects were amplified by the nested PCR method for HBV DNA. using QIAamp DNA Blood Mini, according to the kit operation instructions HBV DNA was extracted from the serum of 200 mu L, which was used as a template to design and synthesize 2 pairs of primers to amplify HBV S gene. The amplified product was identified by 1% agarose gel electrophoresis and purified and recovered by Wizard SV Gel and PCR Clean-Up System kit. The ASTAR software compares the nucleotide sequence of the sequenced specimens with the standard sequence of HBV A-H genotypes obtained from the GenBank, draws the gene evolution tree and determines the genotype. The amino acid sequence is deduced from the nucleotide sequence and the serum subtype is determined. The statistical processing score of the exact probability method of Fisher in the experimental fruit is carried out by the SAS 8.1 software. In the case of P0.05, there is a statistical significance.
Results: of the 110 serum specimens, 97 cases of HBV DNA were positive, and the positive rate was the genotype distribution of 88.2%.97 samples: 9 cases of B genotype, 9.3% in C type, 89.7% in C type and 1 in D type, which accounted for 77 cases of adr subtype, 79.4%; adw2 subtype 11, Ayr subtype 8, 8.2%; ayw2 subtype 8.2%; ayw2 subtype 1 cases accounted for 1.0%.9 cases genetic specimens Serum subtypes were adw2 subtypes; serum subtypes of 87 C genotypes were adrq+ 75 cases (86.2%), 2 cases (2.3%), adw2 2 cases (2.3%), Ayr 8 cases (9.2%), and 1 D genotypes of serum subtype ayw2 subtype. The serum subtypes of different genotypes were statistically analyzed with Fisher exact probability method, P value 0. There were P values 0. There were P values. Statistical significance.
Among the 9 cases of B genotype infection, 4 cases (44.4%) were ASC and 5 were CH (55.6%). 87 cases of C genotype infection, 3 were restorer (3.4%), 73 were ASC (83.9%), 7 was CH (8.1%), LC (3.4%) and HCC were HCC (ASC), and ASC (ASC).Fisher probability method for the clinical outcome of different genotypes, P values were 0.0288, there is a statistical significance.
A total of 84 HBV S gene 540nt sequences were obtained in this experiment. By analyzing the nucleotide substitutions of these nucleotide sequences and the mutation sites of amino acid sequences derived from nucleotide sequences, 131 sites were found to have basic substitutions, of which 40 were synonymous, 89 were missense mutations and 2 loci were used. The main structure homologous region (MHR, aa100-160) found that 45 sites have base substitutions, 18 of which are synonymous and 27 are missense. 2 sites in the missense mutation produce significant amino acid mutations - Q129H (1 cases) and M133L (1 cases), and.1 in the first stem ring structure of the antigen determinant a in a There were three amino acid TTE insertions between a119 and 120. The frequency of HBV S gene substitution was 2.19 per 100 nucleotides.
Conclusion:
The distribution of HBV genotypes in Zhaodong City of Heilongjiang Province was mainly C genotype, with a small number of B and D genotypes.
2 The distribution of HBV serotypes in Zhaodong City of Heilongjiang Province was mainly ADR subtypes (adrq + and adrq - both, but mainly adrq +), with a small number of adw2, Ayr and ayw2 subtypes.
The serum subtypes of different genotypes of different genotypes of 3 HBV were all adw2 in the serum subtypes of.B genotypes; the serum subtypes of C specimens were mainly ADR, a small amount of adw2 and Ayr; the serum subtypes of D type specimens were ayw2. suggesting that there was a certain correlation between the HBV genotypes and the serum subtypes in the D type specimens.
The clinical outcomes of 4 HBV genotypes were both ASC and CH in different.B genotypes. There were a little more.C genotypes in CH and ASC than those with ASC, and the second was the recovery. LC and HCC. suggested that the B genotypes were easier to form.
The 5 HBV S gene found base substitution at many sites, but the near 1/3 base was replaced by a synonymous mutation. There was a certain HBV S gene mutation in the natural population, and no vaccine related mutations were found. The average frequency of the nucleotide substitution was low, suggesting that the S gene was rather conservative.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R181.3

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6 梁雪松;IRES特異性抑制性RNA阻斷丙型肝炎病毒基因表達(dá)的研究[D];中國(guó)人民解放軍第四軍醫(yī)大學(xué);2003年

7 毛紅霞;丙型肝炎病毒不同基因亞型NS3蛋白酶比較及相關(guān)細(xì)胞蛋白的篩選[D];復(fù)旦大學(xué);2003年

8 王怡;RDA技術(shù)分析病毒性肝硬化、原發(fā)性肝細(xì)胞癌相關(guān)基因的研究[D];中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院;2004年

9 王剛;HBV感染HepG2細(xì)胞早期過(guò)程研究[D];山東大學(xué);2005年

10 胡衛(wèi)江;庚型肝炎病毒轉(zhuǎn)基因小鼠模型的建立及其特性研究[D];第二軍醫(yī)大學(xué);2001年

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1 關(guān)嵐;男性吸毒人群中丙型肝炎病毒感染狀況及其影響因素的研究[D];中南大學(xué);2003年

2 蘇小平;乙型肝炎病毒preS2-S基因（adr亞型）轉(zhuǎn)基因小鼠的建立[D];第二軍醫(yī)大學(xué);2003年

3 葛軍輝;乙型肝炎病毒變異型s基因疫苗逆轉(zhuǎn)轉(zhuǎn)基因小鼠免疫耐受的研究[D];第二軍醫(yī)大學(xué);2003年

4 賀啟貴;門(mén)脈高壓性胃病病理初步分析[D];山西醫(yī)科大學(xué);2003年

5 韓香子;延邊地區(qū)庚型肝炎病毒感染狀況的研究[D];延邊大學(xué);2001年

6 張勝權(quán);N-乙酰半胱氨酸(NAC)抗乙肝病毒(HBV)作用[D];安徽醫(yī)科大學(xué);2001年

7 趙書(shū)民;乙型肝炎病毒pres_2-s基因（ayw亞型）轉(zhuǎn)基因小鼠的建立[D];第二軍醫(yī)大學(xué);2002年

8 孫利;丙型肝炎病毒DNA疫苗的實(shí)驗(yàn)研究[D];中國(guó)人民解放軍第四軍醫(yī)大學(xué);2003年

9 彭翼飛;限制性顯示PCR技術(shù)在丙型肝炎病毒基因芯片制備中的應(yīng)用[D];第一軍醫(yī)大學(xué);2001年

10 張偉;乙型肝炎病毒前S區(qū)不同片段的克隆、鑒定及與PreS1基因相互作用蛋白在酵母雙雜交系統(tǒng)中的篩選[D];第四軍醫(yī)大學(xué);2002年

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