CN104480123A - HCV (hepatitis c virus) envelope protein gene and application - Google Patents

Abstract

The invention discloses an HCV (hepatitis c virus) envelope protein gene and an application. The nucleotide sequence of the HCV envelope protein gene is represented as SEQ ID NO.1. RT-PCR (reverse transcription-polymerase chain reaction) and nested PCR technologies are adopted to obtain HCV 1b subtype envelope protein genes popular in China, an expression vector provided with different HCV 1b subtype envelope protein genes is constructed, a two-plasmid cotransfection method is adopted to establish a pseudovirus database, high-infectivity pseudovirion is screened out, and an effective model is provided for studying screening of anti-HCV medicine and immune effect evaluation of HCV vaccines, meanwhile, early characteristics of HCV infection can be further studied, and a technology platform can be provided for evaluation of response of a serum neutralizing antibody from an HCV patient and an antibody of an experimental vaccine and the like.

Description

A kind of HCV envelope protein gene and application

Technical field

The invention belongs to technical field of bioengineering, relate to a kind of HCV envelope protein gene and application.

Background technology

About there are 1.7 hundred million chronic hcv infection person and patients in the current whole world, accounts for 3% of population in the world.Wherein about there are 4,000 ten thousand the infecteds in China.The maximum feature of hepatitis C is that slow-speed rate is very high, in the middle of acute infection person, about 80% the infected can be transformed into chronic hepatitis, wherein about 20% chronic hepatitis patient can develop into liver cirrhosis, have again 1%-4% people finally can develop into liver cancer in patient with liver cirrhosis, the whole world about has 1.7 hundred million patients with chronic hepatitis Cs to be faced with the risk that liver cirrhosis and liver cancer occur at present thus.Worldwide high infection rate, height slow-speed rate and treatment means, vaccine shortage make HCV become the focus of research.

The same with other RNA viruses, HCV has one to have the RNA polymerase relied on the RNA lacking correct functioning compared with poor replication fidelity.So HCV has the variability of height, or even between the different isolates of the same share serum of same patient, just there is the difference of nucleotide sequence or aminoacid sequence.In fact the virus in each patients serum is made up of gene order very high homology, the virus groups that is made up of the virus of minute differences again, and this feature of HCV is exactly quasispecies.So existing 7 the evolution branches (1,2,3,4,5,6,7) of HCV comprise 11 genotype (1a, 1b, 2a, 3a, 3b, 4a, 5a, 6a etc.) and more than 100 gene hypotype at present.HCV genotype and hypotype also exist comparative prevalence difference between regions of the world.China mainland Major Epidemic be have 1b, 2a, 6a tri-type, based on 1b type, the ratio of 6a hypotype had the trend of rising in recent years, mainly common in AIDS patient and drug addict crowd.

The mankind and chimpanzee are the natural hosts of HCV, still lack small animal model effectively now, and cell cultures titre is low, so pseudovirus technology has just shown its plurality of advantages.Strong for infectivity, pathogenic height, the HCV virus of not easily cultivating, pseudovirus technology is a kind of effective comparatively safe research means.

HCV full-length genome is about 9600bp, and encode 4 kinds of structural protein (core protein C, envelope protein E1, E2, p7) and 6 kinds of Nonstructural Proteins are (NS2, NS3, NS4a, NS4b, NS5a, NS5b) respectively.Wherein envelope protein E1 E2 is the chief component on hepatitis C virus surface.They are positions that HCV directly contacts with immunity system or liver cell (main target cell) surface of external environment, host.There is many epi-positions in E1E2 albumen, especially induce the epitope of neutralizing antibody to be sitting at the hypervariable region of E2.Due to the high mutation rate of HCV envelope protein gene, so specific pseudovirus type should be adopted to carry out neutralizing antibody detection to the patients serum that different areas different crowd is originated.But the pseudovirus of bibliographical information at present, its envelope protein gene is mainly derived from type strain sequence, and detection specificity exists limitation, and pseudovirus packaging efficiency is low, can not meet the subsequent experimental such as neutralizing antibody evaluation (vaccine evaluation).In addition, there is notable difference in the pseudovirus packaging efficiency of different subtype, and current research data display 5a is best, and 1b, 2a, 6a are the poorest; And China popular be 1b, 2a and 6a just.Therefore, set up the HCV pseudovirus storehouse of infecting for population of China to have very important significance.

Summary of the invention

The object of the invention is to the above-mentioned defect overcoming existence of the prior art, a kind of HCV envelope protein gene and application are provided.Be specially:

1, the pseudovirus based on HCV Chinese pathogenic strain different subtype is set up, for HCV infection early property (function of envelope protein, the potential acceptor of cell entry cell and hepatitis C virus pseudovirus host preferendum) research, neutralizing antibody level and experimental vaccine Efficacy evaluation provide valid model.

2, the infectious pseudovirion of the height of results, can be used for the evaluation of the in-vitro screening of antiviral, the evaluation of immune effect of vaccine and neutralizing antibody, and then provides support for HCV prevents and treats.

Its concrete technical scheme is:

A kind of HCV envelope protein gene, its nucleotide sequence is as shown in SEQ ID NO:1.

HCV envelope protein gene of the present invention is packed at HCV pseudovirus, the function of research HCV envelope protein, the potential acceptor of cell entry cell and hepatitis C virus pseudovirus host preferendum, and the antibody response etc. evaluating the neutralizing antibody and experimental vaccine that come from hepatitis C patients serum study in application.

Compared with prior art, beneficial effect of the present invention is:

The present invention adopts RT-PCR, sleeve type PCR technology, obtain the HCV1b hypotype envelope protein gene that China is popular, build the expression vector with the different membrane-associated protein gene of HCV1b hypotype, double-mass model cotransfection method establishment pseudovirus storehouse, filter out high infectious pseudovirion, and then for studying the screening of anti-HCV medicament and HCV vaccine Efficacy evaluation provides valid model.Simultaneously also can study HCV infection early property (function of envelope protein, the potential acceptor of cell entry cell and hepatitis C virus pseudovirus host preferendum etc.) further, and evaluate the antibody response etc. coming from hepatitis C patients serum neutralizing antibody and experimental vaccine technology platform is provided.

Accompanying drawing explanation

Fig. 1 is technical solution of the present invention schematic diagram;

Fig. 2 is relative luciferase activity value comparative result figure, and wherein 366 is HCV-1b hypotype numbering, and St is the strain of 1b international standard, and Hebei is the strain of 1b Chinese Industrial Standards (CIS);

Fig. 3 is that Westernblot identifies envelope protein E 2 protein expression;

Fig. 4 is that Westernblot identifies pseudovirus skelemin P24 protein expression.

Embodiment

Below in conjunction with the drawings and specific embodiments, technical scheme of the present invention is described in more detail.

With reference to Fig. 1, technical scheme of the present invention is specially:

1, use lS reagent extracts HCV nucleic acid from HCV infection person's serum;

2, the HCV different genes hypotype envelope protein encoding gene of serum origin cloned and sequenced and design universal primer;

3, RT-PCR accurate amplification goal gene HCV-CE1E2;

4, the glue of goal gene HCV-CE1E2 obtains tens clones after reclaiming purifying and TA clone;

5, carry out enzyme respectively and cut back to close fragment, connect pVRC expression vector, conversion, upgrading grain, obtain pVRC-366-CE1E2 expression plasmid multiple;

6, pVRC-366-CE1E2 and packaging plasmid pNL4-3.Luc.R-E, uses non-liposomal transfection reagent hD cotransfection 293FT cell, packs out tens pseudoviruss with HCV envelope protein;

7, the pseudovirus packed out goes to infect Huh7.5-CD81 (liver cancer cell), judges efficiency of infection by the detection of uciferase activity.

This experiment completes at CDC.Filter out HCV 1b hypotype envelope protein pseudovirion target cell to high infection titer, and by testing proof as follows.

The result of infection experiment several times of No. 1.366 HCV 1b hypotype pseudoviruss is as shown in table 1:

Table 1

	1	2	3	6	7	8	10	11	12	13	14	15	16
														For the first time	-	(+++)	-	-	-	-	-	(++)	(++)	-	-	(+++)	-
For the second time	-	(++)	-	-	-	-	-	(++)	-	-	-	(++)	-
														For the third time	-	(+++)	+	(+)	+	(+)	(+)	(++)	(++)	(+)	(+)	(++)	+
	17	18	19	20	21	22	23	24	25	26	29	32	34
														For the first time	-	-	-	-	(++)	-	-	-	+	(++)	+	-	-
For the second time	-	-	-	+	-	-	-	-	+	(++)	-	-	-
														For the third time	(+)	(+)	(+)	+	(++)	(+)	(+)	-	(++)	(+++)	+	+	-
	36	39	42	43	44	45	46	47	48	49	50	51
														For the first time	+	+	-	-	+	+	-	(++)	-	-	+	-
For the second time	-	-	-	-	-	-	-	+	-	-	-	-
														For the third time	+	+	(+)	(+)	-	(+)	-	(+++)	-	(+)	-	-

2. relative luciferase activity value more as shown in Figure 2.

3.Westernblot identifies envelope protein E 2 and pseudovirus skelemin P24 protein expression as Figure 3-Figure 4.

The above; be only the present invention's preferably embodiment; protection scope of the present invention is not limited thereto; anyly be familiar with those skilled in the art in the technical scope that the present invention discloses, the simple change of the technical scheme that can obtain apparently or equivalence are replaced and are all fallen within the scope of protection of the present invention.

Claims (2)

1. a HCV envelope protein gene, is characterized in that, its nucleotide sequence is as shown in SEQ ID NO:1.

2. described in claim 1, described HCV envelope protein gene is packed at HCV pseudovirus, the function of research HCV envelope protein, the potential acceptor of cell entry cell and hepatitis C virus pseudovirus host preferendum, and the antibody response evaluating the neutralizing antibody and experimental vaccine that come from hepatitis C patients serum study in application.